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6.19. Appendix 19. Substances included in the "EU Pesticides Database" as of April 21
2009. ................................................................................................................................. 224
6.20. Appendix 20. Communication at the 12th
meeting of the Working Group " Insect
Pathogens and Insect Parasitic Nematodes" of IOBC-wprs, in Pamplona (Spain)
22-25 June 2009. .................................................................................................................. 232

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List of tables
Table 1: Number of times that review papers reported research on different
CBC practices...................................................................................................18
Table 2: Number of times that review papers reported research on different
CBC practices in different crop types. ..............................................................19
Table 3: Relationship between pest order and the class of natural enemy
addressed by the CBC research reported ........................................................19
Table 4: Experimental systems used for the study of different CBC practices or
techniques ........................................................................................................20
Table 5: The frequencies that reports of CBC techniques were accompanied
by assessments of effects on natural enemies and pests. ...............................21
Table 6: Representation of European and other countries in reported CBC
research............................................................................................................25
Table 7: Focus of CBC research in Europe and elsewhere: (a) CBC practice or
technique ..........................................................................................................26
Table 8: Focus of CBC research in Europe and elsewhere: (b) crop type......................26
Table 9: Focus of CBC research in Europe and elsewhere: (c) experimental
system ..............................................................................................................27
Table 10: Representation of European and other institutions in authorship of the
reviews that were the source literature for this meta-review.............................28
Table 11: Scientific papers published between 1973 and 2008 on biological
control against major plant diseases (from CAB Abstracts® database). ..........42
Table 12: Numbers of references on biocontrol examined per group of
disease/plant pathogen.....................................................................................43
Table 13: Numbers of different biocontrol compounds and microbial species
reported as having successful effect against key airborne
pathogens/diseases of selected crops. Detailed information and
associated bibliographic references are presented in Appendices 10-
14......................................................................................................................44
Table 14: Microbial species of fungi/oomycetes, yeasts and bacteria reported to
have a significant effect against five main types of airborne diseases
or pathogens in laboratory conditions or in the field (yellow highlight).
Bibliographic references are presented in Appendices 10 to 14. .....................45
Table 15: References extracted from the CAB Abstracts database and
examined for reviewing augmentation biological control in grapevine..............52
Table 16: Biocontrol agents evaluated in researches on augmentative biological
control of pests in grapevine.............................................................................54
Table 17: Number of references on augmentative biocontrol agents per group
and species of target pest in grapevine. ...........................................................55
Table 18: Number of references reporting data on the efficacy of augmentative
biocontrol of pests in grapevine. .......................................................................57
Table 19: Recent introductions of parasitoids as Classical Biocontrol agents..................69
Table 20: Consulted sources of information on authorized biocontrol plant
protection products in five European countries:................................................75
Table 21: Active substances suitable for biological control listed on Annex I of
91/414/EEC (EU Pesticide Database) - Status on 21st April 2009...................76

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Table 22: Evidence for, and effectiveness of, induced resistance in plants by
Trichoderma species (Harman et al., 2004a). ..................................................86
Table 23: Trichoderma-based preparations commercialized for biological control
of plant diseases...............................................................................................88
Table 24: Compared structure of the production costs for a microbial biocontrol
agent (MBCA) and a chemical insecticide (source IBMA). ...............................98
Table 25: Compared potential costs of registration for a microbial biocontrol
agent (MBCA) and a chemical pesticide (source IBMA)...................................99
Table 26: Compared estimated market potential for a microbial biocontrol agent
(MBCA) and for a chemical pesticide (source: IBMA).......................................99
Table 27: Compared margin structure estimates for the production and sales of
a microbial biocontrol agent (MBCA) and a chemical pesticide (source
IBMA)..............................................................................................................100
Table 28: Production systems selected for a survey of factors influencing
biocontrol use in Europe (source IBMA) .........................................................103
Table 29: Geographical distribution of sampling sites for a survey of factors
influencing biocontrol use in Europe (source IBMA).......................................103
Table 30: Structure of the questionnaire used in a survey of European farmers
and retailers of biological control products .....................................................103
Table 31: Impact of twelve factors on the future use of biocontrol agents by
European farmers according to a survey of 320 farmers................................106

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List of Figures
Figure 1: Year of publication of review papers analysed .................................................16
Figure 2: Reported influence of CBC on natural enemies, pest control and intra-
guild predation (IGP).........................................................................................22
Figure 3: Reported influence of different CBC practice and technique groups on
abundance or fitness of natural enemies. See Fig. 2 for key to colours. ..........22
Figure 4: Reported influence of different CBC practice and technique groups on
pest control. See Fig. 2 for key to colours. .......................................................23
Figure 5: Reported effectiveness of CBC in promoting the abundance or fitness
of natural enemies in different crop types. See Fig. 2 for key to
colours. .............................................................................................................24
Figure 6: Reported effectiveness of CBC in pest control in different crop types.
See Fig. 2 for key to colours. See Fig. 2 for key to colours ..............................24
Figure 7: Numbers of review papers recommending further research in different
‘practice or technique’ groups...........................................................................29
Figure 8: Categories of science underpinning CBC that were identified by
review papers to need further research. ...........................................................30
Figure 9: Categories and sub-categories of science underpinning CBC that
were identified by review papers as needing further research..........................31
Figure 10: Categories of challenge to the implementation of CBC that were
identified by review papers. ..............................................................................32
Figure 11: Categories and sub-categories of challenge to the implementation of
CBC that were identified by review papers.......................................................33
Figure 12: Evolution of the yearly number of publications dedicated to biological
control of plant diseases based on a survey of the CAB Abstracts®
database...........................................................................................................41
Figure 13: Range of efficacy of 157 microbial biocontrol agents against five main
types of airborne diseases. Detailed data are presented in.............................50
Figure 14: Number of papers per year published during 1998-2008 concerning
augmentative biological control of pests in grapevine. .....................................54
Figure 15: Groups of biocontrol agents investigated in augmentative biological
control researches in grapevine. Number of references for each group
is reported.........................................................................................................58
Figure 16: Groups of target pests investigated in augmentative biological control
researches in grapevine. Number of references for each group is
reported. ...........................................................................................................58
Figure 17: Large-scale temporal survey of the publications associated with
classical biological control ................................................................................64
Figure 18: Relative importance of the different types of biocontrol during the
temporal frame [1999-2008] .............................................................................65
Figure 19: Number of pest species and related citation rate by orders during the
period [1999 ; 2008]..........................................................................................66
Figure 20: Relationships between the number of publications associated to the
main pests and the relative percentage of ClBC related studies. .....................67
Figure 21: Frequencies of papers and associated median IF related to the
different categories of work...............................................................................68
Figure 22: Estimated sales of biocontrol products in Europe in 2008 (in Million €).
The estimates were obtained by extrapolating use patterns in a
representative sample of EU farmers. ............................................................104
Figure 23: Estimated distribution of biocontrol use among types of crops in 2008
in Europe ........................................................................................................104

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Summary
Research Activity 4.3 of ENDURE has brought together representatives of industry and
scientists from several European countries with experience ranging from fundamental biology
to applied field work on biological control against pests and diseases. The unique diversity of
expertise and concerns allowed the group to set up very complementary approaches to
tackle the issue of the factors of success of biocontrol.
The initial part of the work accomplished by this group consisted in a thorough review of
scientific literature published on all types of biological control. Although it had to be focused
on selected key European crops and their major pests and pathogens, this review is unique
in the scope of the topics it covered and in the comprehensive inventories it allowed to gather
on the potential of biocontrol and factors of success at field level. A large part of this study
was dedicated to the increasingly promising field of research on conservation biological
control.
In parallel with identifying knowledge gaps and key factors from published research,
information was gathered on aspects linked to the production and commercialization of
biocontrol agents.
These results, complemented by the views of experts in the field of biocontrol consulted at
the occasion of meetings of IOBC-wprs, allowed the identification of majors gaps in
knowledge and bottlenecks for the successful deployment of biocontrol and lead to the
proposition of key issues for future work by the research community, the field of development
and prospects for technological improvement by industry.

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Definitions
Biological control / biocontrol
Many different definitions of biological control have been proposed. Most of them are clearly
related to the control of pests, mainly insects and mites, and have been extrapolated to
disease control. Eilenberg (2006) defined “biological control or biocontrol as the use of living
organisms to suppress the population density or impact of a specific pest organism, making it
less abundant or less damaging than it would otherwise be”.
Augmentation / augmentative biological control
Augmentation biological control includes activities in which natural enemy populations are
increased through mass culture, periodic release (either inoculative or inundative) and
colonization, for suppression of native or non-native pests. (Orr, 20091
)
Classical (also "importation") biological control
Classical biological control is defined in this study as the intentional introduction of an exotic,
usually co-evolved, biological control agent for permanent establishment and long-term pest
control.
Conservation biological control
“Conservation biological control involves manipulation of the environment to enhance the
survival, fecundity, longevity, and behaviour of natural enemies to increase their
effectiveness” (Landis et al., 2000). See also chapter 3.1.
Microbials / Micro-organisms used for biological control.
The term micro-organism is defined in Council Directive 91/414/EEC (as amended by
Commission Directive 2001/36/EC) as follows: A microbiological entity, cellular or non-
cellular, capable of replication or of transferring genetic material. The definition applies to, but
is not limited to, bacteria, fungi, protozoa, viruses and viroids. It does not include multicellular
organisms, such as nematodes or insects.
Pest
Unless otherwise specified, the term "pest" in the present document is meant to cover the
whole range of crop-damaging animals (invertebrate and vertebrate), agents of plant
diseases and weeds.
1
Orr, D. 2009. Biological Control and Integrated Pest Management, pp 207-239 IN: R. Peshin, A.K. Dhawan
(eds.), Integrated Pest Management: Innovation-Development

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1. Foreword
The present document results from the collective effort of researchers from five European
research institutions (CNR, INRA, RRes, UdL and WUR), together with representatives of
the International Biocontrol Manufacturers Association (IBMA)
The organisation and edition of this document was coordinated by P. NICOT (INRA-Avignon).
The specific contributors to the research work and their role / expertise were as follows:
ALABOUVETTE Claude, INRA, Plant Pathologist (review of scientific literature on
biocontrol against Fusarium; analysis of regulatory issues; review of CBC of soil-
borne pathogens)
ALOMAR Oscar, UdL, Entomologist (meta-review of scientific literature on conservation
biological control against arthropod pests)
BARDIN Marc, INRA, Plant Pathologist (review of scientific literature on biocontrol
against Botrytis, rusts)
BLUM Bernard, IBMA (economic survey, regulatory issues)
FERGUSON Andrew, RRES, Entomologist, (meta-review of scientific literature on
conservation biological control against arthropod pests, with assistance from Stephen
Moss and Jonathan Storkey for section on weed control)
GIORGINI Massimo, CNR, Entomologist (review of classical and augmentative/inundative
biocontrol against arthropod pests)
HEILIG Ulf, IBMA (inventory of commercial biocontrol products, and their usage in 5
European countries; analysis of regulatory issues)
KÖHL Jürgen, WUR, Plant Pathologist (Venturia, Ulocladium)
MALAUSA Jean Claude, INRA, Entomologist (review of scientific literature on classical
biocontrol and parasitoids against arthropod pests)
NICOT Philippe, INRA, Plant Pathologist, (review of scientific literature on biocontrol
against downy mildews and late blight, Monilia; analysis of reviews concerning plant
diseases; review of CBC of aerial pathogens)
RIS Nicolas, INRA, Entomologist (review of scientific literature on classical biocontrol
and parasitoids against arthropod pests)
RUOCCO Michelina, CNR, Plant Pathologist (review of scientific literature on biocontrol
against downy mildews; review of factors of efficacy of biocontrol agents against
plant diseases)
2. Introduction
Biological control methods against pests and diseases constitute key elements for the
development of integrated protection and integrated production of cultivated crops.
The objectives of the present study were to conduct a review of the current status of
European research on the exploitation of natural biological processes for Biological control,
to identify knowledge gaps and the main constraints for its implementation in the field and
finally, to provide suggestions for possible improvements and needs for further research
efforts.
In this study, both conservation biological control and the use of classical or augmentative
biological control have been considered.

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To optimize the available expertise and human resources gathered for this Research
Activity, efforts were focused on (but not systematically limited to) situations relevant for
Case Studies and Systemic Case Studies conducted elsewhere by the ENDURE network.
The targets of the biological control were mostly arthropod pests and plant pathogens.
Biological control of weeds was only marginally considered due to the lack of weed experts
participating in the group.
3. Conservation biological control (CBC): current status of
research relevant to the major cropping systems in Europe
and recommendations for multi-site experiments
Contributors:
Andrew Ferguson RRES, Entomologist, field crops, co-leader, with assistance from
Stephen Moss and Jonathan Storkey for section on weed control
Oscar Alomar UdL, Entomologist, field vegetables, co-leader
Claude Alabouvette INRA, Pathologist, soil pathogens
Philippe Nicot INRA, Pathologist, aerial pathogens
3.1.Definitions related to Conservation Biological Control
“Conservation biological control involves manipulation of the environment to enhance the
survival, fecundity, longevity, and behaviour of natural enemies to increase their
effectiveness” (Landis et al., 2000)
“Modification of the environment or existing practices to protect and enhance specific natural
enemies or other organisms to reduce the effect of pests” (Eilenberg et al., 2001)
This encompasses:
 protection and/or enhancement of natural enemies or other naturally-occurring
organisms that reduce the effect of pests by manipulating their environment and
providing resources to increase their effectiveness.
For the purpose of this review, this does not encompass:
 released biological control agents.
 cultural control measures that depend for success on their direct effects on pests
rather than on protection or enhancement of natural enemies or other non-pest
organisms.
In this review the term ‘natural enemy’ (NE) is preferred over ‘biological control agent’ for
describing organisms with a potential role in CBC. ‘Natural enemy’ is a more inclusive term
that can be applied to any organism with a trophic relationship with a pest that has potential
value for the biocontrol of that pest. By contrast, the term ‘biological control agent’ implies a
proven ability to control the pest.
3.2.Scope and aims of this chapter
Pest groups considered:
 Invertebrate pests, the main focus of the report
 Plant pathogens, airborne and soil-borne
 Weeds
The main focus of this report is on CBC for the management of invertebrate pests. This is
the field for which the terminology was coined and in which it is most used. However, the

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authors believe that the principles of CBC could be applied equally to management of plant
pathogens and weeds should the appropriate circumstances exist for conserving and
promoting their natural enemies. Therefore separate sections of this report give
consideration to research relevant to CBC of airborne plant pathogens, soil-borne plant
pathogens and weeds.
Reviews of research literature:
Each pest group is the subject of review of research literature to:
 establish the status of CBC research relevant to the major cropping systems of
Europe and
 identify gaps in the scientific knowledge-base that represent impediments to the
implementation of CBC and require further research.
The review of invertebrate pests also reports on other challenges to CBC implementation
identified by authors of review papers.
Recommendations for multi-site experiments:
Priorities for future research in European cropping systems that would benefit from a multi-
site, supra-national approach are identified from the reviews (see summary). It is proposed
that these priorities should be discussed at a joint workshop of ENDURE research sub-
activities 4.3 (biocontrol), systems case studies and 2.3 (landscape), for consideration of
gaps in research and joint recommendations for multi-site experiments. The ENDURE
Annual Meeting in October 2009 in Wageningen would be a suitable occasion for this.
3.3.Limits of this report
 This report does not attempt to cover CBC in every crop and cropping system. Its
focus is determined by what authors of the review literature consider to be the most
important research results and knowledge gaps.
 This report does not review the value for CBC of chemical pesticides that are
selective in their action, i.e. less injurious to biological control agents than to pests.
This principle is well accepted.
 This report does not address regulatory or policy issues that influence the uptake of
CBC except inasmuch as they are mentioned in the papers reviewed.
 This report does not attempt to survey the current extent of implementation of CBC.
3.4.Reviewing methods
 Invertebrate pests: a meta-review of the review literature published between 1989
and 2009.
 Plant pathogens expert knowledge of the authors and their colleagues, citing the
 Weeds most significant primary and review literature.
Invertebrate pest literature review
 A meta-review of peer-reviewed published research on CBC relevant to the major
cropping systems in Europe since 1989.
 This is accomplished primarily by examination and analysis of review papers
identified in a search of Web of Science, CAB Abstracts and BIOSIS databases.
 Search terms:
Literature searches for reviews were based on the following search terms:
- conservation or “habitat management” or “ecological infrastructure*”
and
- biocontrol or “biological control” or “natural enem*” or predator* or parasitoid*

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A number of additional review references identified during the course of this study
were added as the search did not identify all relevant review papers, particularly
those published as book chapters.
 Spatial scale: this review focuses on field to landscape scales, i.e.:
o The sown crop
o The field margin (components and terminology as Greaves and Marshall, 1987
and Marshall and Moonen, 2002).
o Managed non-crop habitats within the field (e.g. beetle banks, flower margins etc)
o Landscape scales (within c. 2-3km radius).
Note that landscape issues are also addressed in depth by ENDURE RA2.3 and the
impact of landscape management on pest abundance is the subject of RA 2.3
Deliverable DR 2.9, ‘Synthesis on impacts of landscape characteristics on densities
of pests and their regulation by natural enemies’. As landscape management is of
such great importance in the management of natural enemies, no comprehensive
review of CBC would be complete without its inclusion. The RA 4.3 and RA 2.3
reviews will provide complimentary views of the role of the landscape in pest
management. The present RA 4.3 review is a meta-review assessing landscape
management in the wider context of all CBC techniques whereas the RA 2.3 review
assesses the primary research literature and provides is focussed solely on
landscape issues.
 The geographic coverage of the review is world-wide but is focussed on studies
relevant to the major cropping systems in Europe
 The following are reviewed:
- major research results, CBC techniques and issues discussed in the review
literature whenever the subject is relevant to the European situation
- CBC techniques or practices that have potential for practical application or are
already practically applied
- CBC research reported in relation to crop type, natural enemy taxon and pest
taxon.
- experimental systems used in CBC research
- evidence for the success of different CBC techniques in supporting natural
enemies and depressing pest populations
- evidence for the success of CBC in different cropping systems
- the strength of the contribution of European scientific institutions to the primary
literature on CBC research reported in review papers and the relationship of that
contribution with crop type and CBC technique.
- the relative contributions of scientific institutions in Europe and elsewhere to the
authorship of review literature concerning CBC
- analysis of the number of review papers addressing CBC research by year, 1990-
2009
- gaps in the scientific knowledge-base that represent impediments to the
implementation of CBC and require further research.
- other challenges to CBC implementation identified by authors of review papers.
 Scoring of content of review papers
Each review paper was scored on a spreadsheet using the following headings:
A. Headings scored for each aspect of CBC research discussed by each review
paper, including efficacy of CBC techniques (each combination of headings 1-13
that was unique within each review paper was separately scored; thus for the
same review paper there could be several lines of data):
1. crop type
2. experimental system
3. whether research includes modelling
4. the country where experiments were done

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5. whether experiments were done in Europe
6. CBC practice and techniques group
7. specific CBC practice or technique
8. pest species or group
9. class of natural enemy
10. natural enemy species or group
11. effect on abundance or fitness of natural enemy
12. effect on pest control
13. effect on intra-guild predation (IGP)
B. Headings for the views of review paper authors on gaps and challenges
14. research gaps identified
15. challenges to implementation discussed
C. Headings for details of authors and institutions
16. reference number
17. year published
18. first author
19. any authors based in Europe?
20. country or countries where authors' institution(s) located
21. names of authors' institution(s)
The content of the review paper was scored under each heading by allocation of one
of a number of alternative terms. A complete table of headings and of the terms that
could be used under each heading when scoring the papers is given in Appendix 1.
Note that terms are scored under ‘CBC practices and techniques’ headings when
research that supports the development of those techniques is reported. For
example, a score under ‘Landscape management’ does not necessarily imply that the
landscape was manipulated but that data were collected that would assist the future
design and management of landscapes to optimise CBC.
 Data analysis and collation
 Frequencies of occurrence of different terms under headings relating to aspects
of the CBC research reported were summarised simply, using XL spreadsheets
to draw up two-way tables and bar charts. Particular attention was paid to:
- the relationship between choice of CBC techniques and practices and the target
pest, the cropping system and the experimental system
- the effectiveness of CBC in relation to CBC technique and cropping system
- the contribution of Europe to CBC research in relation to CBC technique,
cropping system and experimental system
 The reporting of CBC research gaps and of challenges to CBC implementation
was analysed by grouping them into categories to enable their collation and
analysis of frequencies using bar charts.
Research gaps identified were subjected to a two-part analysis:
1. Analysis of gaps in relation to particular CBC practices and techniques groups:
The aim of the first analysis was to enable a direct comparison of the weight
given by reviews to the reporting of different CBC techniques (from past
research papers) with the needs for future research identified by the reviews.
Gaps were allocated (where possible) to one of six headings matching the
categories of CBC techniques by which past research was analysed: ‘limiting
pesticide use’, ‘manipulation of behaviour’, ‘reduced disturbance’, ‘provision of
refugia and resources’, ‘increased biodiversity’ and ‘landscape management’.
2. Analysis of gaps in the science underpinning CBC:

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The second analysis was more comprehensive and was designed to
summarise the gaps in science that underpins CBC according to scientific
discipline. It covered all gaps identified by review papers, not only those
relating to specific CBC techniques. Each reported gap in the science-base
was allocated to one of 11 research gap categories and 36 sub-categories
that were defined a posteriori according to what was found in the review
papers.
The challenges to CBC implementation identified by the review papers were
summarised by allocating each to one of five categories of challenge: scientific
practice, R&D costs, knowledge transfer, socio-economic, and policy. These
categories were further divided into sub-categories.
 The authors, institutions and countries represented in the review literature,
together with their dates of publication, were summarised simply using XL
spreadsheets.
3.5.Results: Management of invertebrate pests
3.5.1. Summary of the source literature for the meta review
Ninety review papers covering CBC research were analysed, see bibliography in Appendix
2. Most of these were published from 1998 onwards (Figure 1). The majority (48) were
published in peer-reviewed international journals; three were published in conference
proceedings and 39 as book chapters. This large body of review literature reflects a large
primary literature. A search of primary papers from 1967 to 2009 in the same databases and
based on the same search terms delivered 2,675 references.
Number of
review papers
per year
Figure 1: Year of publication of review papers analysed
3.5.2. Detailed analysis of CBC research reported by review papers
3.5.2.1. Reporting of CBC methods.
A total of 221 lines of data were scored concerning the reporting of CBC methods.
CBC techniques
 Ten categories and 48 sub-categories of CBC practices or techniques were identified
(Table 1).

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 The four CBC techniques most commonly reported to be the subject of research all
involved the provision or management of resources and refugia in the agroecosystem
for natural enemies, i.e.: the provision of refugia and resources in the field at
concentrated locations (e.g. sown flower strips) or spread across the crop (e.g. weed
management, ground-cover management and mulch), landscape management and
reduced disturbance (reduced tillage) (Table 1). Reports concerning these
techniques made up 177 of all 221 reports (80%).
 Limiting pesticide use through the use of pest resistant crop cultivars (GMO’s or
otherwise), IPM, buffer zones or spatial targeting (precision farming) was much less
discussed (5% of reports).
 Manipulation of (invertebrate) behaviour, habitat manipulation and optimising plant
morphology together comprised 6% of reports.
 Increased ecosystem or natural enemy biodiversity comprised 4% of reports (Table
1).
Crop type
 Arable crops were the subject of more reports of CBC techniques from single crop
types (74%) than any other crop type (Table 2).
 Maize comprised 12% of the reports on arable crops and 9% of all reports from single
crop types.
 Orchards, vines and field vegetables were each the subject of less than 10% of
reports from single crop types (Table 2).
 Perennial crops were the subject of 19% of reports from single crop types.
 Greenhouse vegetables were the subject of only one report, probably because
augmentative biological control is more commonly practised in this system than CBC.
 Provision of refugia and resources was the most commonly reported CBC technique
in each crop type usually followed by landscape management.
Pest taxon
 Hemiptera (mostly aphids) were the target pests in 18% of reports of CBC (Table 3).
Lepidoptera were the next most common targets (6% of reports).
 Thirty percent of the reports of CBC referred to more than one pest.
 A significant proportion (40%) of reports of CBC in the review papers did not specify
the target pest and this was especially true when discussing predators (Table 3).
Natural enemy taxon
 Predators were much the most frequently discussed natural enemies in reports of
CBC (42%; Table 3). Parasitoids were discussed in 17% of reports and
entomopathogenic fungi in 6%.
 A synoptic list of the natural enemy taxa referred to in reviews, and the number of
times they were reported, can be found in Appendix 3. No attempt has been made to
compile a complete list of the natural enemy species referred to.
 Predators were the most commonly discussed natural enemies of hemipteran pests
and parasitoids were the most commonly discussed natural enemies of lepidopteran
and coleopteran pests (Table 3).
 A full analysis of the classes of natural enemy discussed in relation to all pest species
or groups reported can be found in Appendix 4

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Table 4: Experimental systems used for the study of different CBC practices or techniques
CBC practice and techniques
group
Number of times review papers discussed research in different
experimental systems
field lab-
semifield
model
only
various unspecified all
systems
Limiting pesticide use 9 3 12
Manipulation of behaviour 2 1 1 1 5
Habitat manipulation 2 2 1 5
Optimizing plant morphology 1 3 4
Reduced disturbance 13 13
Provision of refugia / resources
at concentrated locations
63 1 1 4 1 70
Provision of refugia / resources
spread across crop
45 2 4 1 52
Landscape management 36 1 2 2 1 42
Increased ecosystem biodiversity 3 1 1 5
Increased biodiversity of NE 1 2 1 4
Various 1 2 1 4
Unspecified 2 3 5
All CBC practices 178 6 3 24 10 221
3.5.2.2. Evidence for the success of different CBC techniques
How the success of CBC was assessed in the literature
 The great majority of reports of CBC techniques were accompanied by an assessment of
the effectiveness of the techniques (Table 5).
 The effect of CBC on natural enemies was discussed in over 90% of reports of CBC
techniques.
 The effect of CBC on pests was discussed much less frequently (47% of reports) and it was
rarely reported without also reporting the effect on natural enemies (Table 5).
 Only reports on the influence of increased ecosystem and natural enemy biodiversity
discussed effects on pests as frequently as they discussed effects on natural enemies
(Table 5).
Over-all effectiveness of CBC
 The implementation of CBC techniques was accompanied by increased abundance or
fitness of natural enemies in the great majority of reports where the evidence was assessed
(94%); the evidence was judged to be strong in 42% of these positive reports (Figure 2).
 CBC was accompanied by increased pest control in 80% of reports where the evidence
was assessed. This evidence was judged to be strong in 25% of those positive reports.
 The effect of CBC on intra-guild predation (IGP) was rarely reported and so will not be
considered further in the results section.

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Table 5: The frequencies that reports of CBC techniques were accompanied by assessments of
effects on natural enemies and pests.
Practice and techniques group Frequency
that effects
of CBC were
not reported
Frequency that effects of CBC
were reported
Total
number
of reportson NE
only
on pest
control
only
on both
NE and
pest
Limiting pesticide use 1 4 7 12
Manipulation of behaviour 1 3 1 5
Habitat manipulation 1 1 3 5
Optimising plant morphology 2 2 4
Reduced disturbance 9 4 13
Provision of refugia / resources
at concentrated locations 3 34 33 70
Provision of refugia / resources
spread across crop 1 25 1 25 52
Landscape management 4 20 1 17 42
Increased ecosystem biodiversity 5 5
Increased biodiversity of natural enemies 1 1 2 4
Various 1 2 1 4
Unspecified 3 1 1 5
All 16 101 3 101 221
Effectiveness of different CBC techniques
 The strongest evidence for a positive effect of CBC on natural enemies was associated with
the three techniques groups covering management of the landscape and provision of
refugia and resources within it (Figure 3). In 99% of the 154 reports of these CBC
techniques there was judged to be accompanying benefit to natural enemies and the
evidence for this was strong in 46% of the reports.
 The other CBC techniques were less reported but most reports were accompanied by
evidence of effects on natural enemies that were exclusively beneficial. There was usually
a smaller proportion of reports where the evidence was judged to be strong except in the
case of ‘increased ecosystem biodiversity’ for which the reported degree of benefit was
similar to refugia and resource provision, probably for the same reasons.
 Evidence for the benefit to natural enemies of limiting pesticide use was surprisingly weak
and this may reflect choice of search terms used rather than the literature published on the
subject. The two reports of negative effects of limiting pesticide use are associated with the
use of insect-resistant GM crops.
 There was no consistent evidence that increasing NE biodiversity of was either beneficial or
detrimental to their abundance or fitness but this subject was reported only twice.
 The strongest evidence for a positive effect of CBC on pest control was again associated
with the three techniques groups covering management of the landscape and provision of
refugia and resources within it (Figure 4). Of the 77 reports discussing effects on pests,
82% reported increased pest control. However, there were fewer reports compared to
natural enemies and the evidence for benefit was strong in 17% of them only.
 Although effects on pest control associated with other CBC techniques were more rarely
discussed, for each technique there were reports of increased pest control and none of
decreased pest control.
 The strong increase in pest control associated with limiting insecticide use was derived from
six reports on the effect of insect-resistant GM crops.

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Key to colours for all coloured bar charts___________
good evidence for a strong increase
some evidence for an increase
no consistent evidence
some evidence for a decrease
good evidence for a strong decrease
 One review reported that there was strong evidence for increased pest control (aphids)
associated with increased natural enemy biodiversity.
 A complete table summarising the reported effects of different CBC practices by category
and sub-category and on pests and natural enemies is given in Appendix 5.
Figure 2: Reported influence of CBC on natural enemies, pest control and intra-guild predation
(IGP).
Figure 3: Reported influence of different CBC practice and technique groups on abundance or
fitness of natural enemies. See Fig. 2 for key to colours.

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Figure 4: Reported influence of different CBC practice and technique groups on pest control. See
Fig. 2 for key to colours.
Effectiveness of CBC in different crop types: natural enemies
 The largest proportion of reports that gave strong evidence that CBC promoted natural
enemies were from field vegetables and vines (63% and 60%, respectively, of reports
where it was assessed) (Figure 5). In 10 of these 12 reports, the strong benefit of CBC to
natural enemies was associated with the provision of refugia or resources and in two
reports (in vines) it was associated with landscape management.
 Many reports relating to arable crops (45%) also linked CBC with strong evidence of natural
enemy promotion. Most such reports concerned the provision of refugia or resources (31
out of 44) or landscape management (8 out of 44).
 Smaller proportions of reports on orchards and maize cited strong evidence for effects of
CBC on natural enemies (29% and 36%, respectively) but each of these reports was again
associated with the provision of refugia or resources.
Effectiveness of CBC in different crop types: pests
 Reports relating to vines had the largest proportion (57%) that referred to strong evidence
that CBC promoted pest control (Figure 6). The CBC techniques associated with this
benefit were the provision of refugia or resources (three reports) and landscape
management (one report).
 For all the other crops, the proportion of reports indicating strong effects of CBC on pest
control was considerably smaller, amounting to a total of ten out of 59 reports (field
vegetables 40%, orchards 33%, arable crops 17%, maize 0%). Five of these ten reports
related to the provision of refugia or resources and one to landscape management. The
remaining four were associated with the use of GM insect-resistant arable crops.

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Figure 5: Reported effectiveness of CBC in promoting the abundance or fitness of natural enemies
in different crop types. See Fig. 2 for key to colours.
Figure 6: Reported effectiveness of CBC in pest control in different crop types. See Fig. 2 for key to
colours. See Fig. 2 for key to colours

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3.5.2.3. The involvement of European research institutions in CBC research
3.5.2.3.1 The involvement of European institutions in the primary research
reported by review papers
Countries represented
 Countries in the European Union have a strong record of research on CBC, being involved
with 63% of all reports of CBC techniques analysed here (Table 6).
 Those reports that derived from individual European countries were exclusively from
northern and central Europe. The UK was particularly well represented in the reports,
followed by Germany and Switzerland.
 The USA was the non-European country most strongly represented in reports of research
derived from a single country, followed by New Zealand (Table 6; note that these countries
were also represented in the ‘EU and elsewhere’ category).
Table 6: Representation of European and other countries in reported CBC research
Region(s) from where
research activity was
reported
Country Number of times
research activity
was reported
Europe: EU Czech Republic 1
Finland 1
Germany 5
Hungary 1
Netherlands 2
Sweden 3
UK 16
various 14
EU and elsewhere 97
Europe: not EU Switzerland 5
All Europe 145
Exclusively outside Europe China 1
New Zealand 6
USA 48
various 4
All 59
Unspecified 17
All regions 221
CBC practices and techniques studied in Europe and elsewhere
 Research on CBC in Europe in the review period focussed strongly on the provision of
refugia and resources (55% of reported research involving any European country),
especially at concentrated locations, and to a lesser extent on landscape management
(17% of reports) (Table 7).
 The focus of CBC research outside Europe was similar, 56% reports concerning the
provision of refugia and resources (but especially those spread across the crop) and 27%
concerning landscape management (Table 7).

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Experimental system used in Europe and elsewhere
 Research on CBC in Europe was overwhelmingly field-based, with only small proportions
relying exclusively on laboratory and semi-field scale studies or on modelling (Table 9).
 Outside Europe, CBC research was also overwhelmingly field-based (Table 9).
 Europe is represented strongly in the record of modelling for CBC research. Modelling was
included in 11 out of 134 studies with European participation (where specified). Modelling
was not reported from the 53 studies exclusively outside Europe where methods and
countries were specified.
Table 9: Focus of CBC research in Europe and elsewhere: (c) experimental system
3.5.2.3.2 The contribution of European institutions to the authorship of the
review papers used for this meta-review
 Institutions in European countries contributed strongly to the review literature on CBC. Half
of all contributing institutions were European and 40% of all contributions were from
European countries (Table 10). For complete lists of contributing institutions in Europe and
elsewhere, together with their contributions to review paper authorship, see Appendices 6
and 7.
 The UK was particularly well represented and the Netherlands and Switzerland also made
strong contributions.
 The USA was the single country most strongly represented in authorship of review papers
(Table 10).
 Countries outside Europe and the USA were represented only by single contributions to
review papers on CBC except for Australia, Kenya and New Zealand. Lincoln University,
the only contributing institution in New Zealand, was involved in a remarkably large number
of the reviews (Table 10; Appendix 7).
Experimental system Number of times review papers reported research activity in:
European
Union
EU and
elsewher
e
Europe
but not
EU
all Europe exclusive
ly outside
Europe
un-
specified
all regions
Field 39 71 5 115 52 11 178
Laboratory - semifield 2 2 4 6
Model only 1 1 2 1 3
Various 1 23 24 24
Unspecified 2 2 3 5 10
All systems 43 97 5 145 59 17 221

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Table 10: Representation of European and other institutions in authorship of the reviews that were
the source literature for this meta-review
Region(s)
where
institutions
were located
Country Number of institutions
contributing to
authorship of review
papers
Total number of times
institutions represented in
authorship of review
papers
Europe: EU Austria 2 2
Belgium 1 1
Denmark 1 2
Finland 1 1
France 2 2
Germany 2 6
Hungary 2 2
Italy 2 1
Netherlands 5 10
Spain 1 1
Sweden 1 2
UK 24 38
Europe: not EU Switzerland 5 7
All Europe 49 75
Outside Europe Australia 5 15
Canada 1 1
Indonesia 1 1
Israel 1 1
Japan 1 1
Kenya 2 3
México 1 1
New Zealand 1 18
USA 37 71
All outside Europe 50 112
All regions 99 187
3.5.3. Knowledge gaps that represent barriers to the implementation of CBC
3.5.3.1. Analysis of gaps in relation to the CBC techniques reviewed in
section 2.5.2
 Landscape management was the CBC practice most frequently identified as a priority for
future research, 39 of the 90 review papers recording this as a need (43%) (Figure 7). This
is proportion is considerably larger than the proportion of reports of past research that were
related to landscape management (19%; Table 5), indicating a view that this area of work
should be expanded.
 The provision of refugia and resources was the CBC practice next most frequently identified
as needing more research (36% of reviews; Figure 7). This topic was the subject of 55% of
the reports of past CBC research in the reviews (Table 5).
 The impact of increased biodiversity on CBC was the subject of only 4% of reports of past
research (Table 5) but was frequently stated as a priority for future research (30% of
reviews; Figure 7).
 Manipulation of behaviour and limiting pesticide use were seen as priorities for further
research by significant minorities of review papers. Reduced disturbance was much less
frequently recommended (Figure 7).

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Figure 7: Numbers of review papers recommending further research in different ‘practice or
technique’ groups.
3.5.3.2. Analysis of gaps in the science underpinning CBC
 The research gap categories identified by review papers were grouped into 11 categories
(Figure 8) and 36 sub-categories (Figure 9). See Appendix 8 for more complete definitions
of the sub-categories.
 ‘Landscape scale interactions’ was the category of gap most frequently identified as a
priority for further research, 41 of 90 review papers recording this as a need (46%) (Figure
8). Within this category, studies of the appropriate spatial scale for landscape management
for CBC and studies of the movement of natural enemies within the landscape were the
topics most frequently recommended for further study (Figure 9).
 Community ecology, autecology and behavioural ecology were identified as priorities for
further research by 35, 23 and 15 review papers (39%, 26% and 17%), respectively. In
these categories the following were considered most important for future study: the impact
of natural enemy diversity, interactions and community dynamics on CBC; the study of the
traits and population dynamics of natural enemies and their responses to habitats; the
manipulation of natural enemy behaviour (e.g. by exploiting chemical ecology, push-pull,
mixed cropping) (Figure 9).
 Determination of plant or habitat characteristics that encourage CBC was recommended as
a priority for further research by 29 out of the 90 review papers (32%) (Figure 8). In this
category the topics considered most important for study included the comparative benefits
of plants and habitats to natural enemies, their management for natural enemies, their role
as sources or sinks for natural enemies, and their relative value to pests and to beneficial
organisms (Figure 9). Only four reviews recommended studies of the influence of plant
resistance to insects on CBC and four recommended studies of the risks and benefits to
CBC of transgenic crops.
 Assessment of the impact of CBC was seen as a priority for future research by 22 review
papers (24%) (Figure 8). It should focus on testing the effectiveness of CBC in relation to
pest control, reduction in pesticide use, improved crop yield and cost-benefit analysis, as
well as identification of natural enemies with the most impact on biological control (Figure 9;
Appendix 6).
 The provision of resources and refugia was a subject considered to need more research by
13 of the reviews (Figure 8) and this should focus on means of managing resources for
natural enemies (e.g. banker plants, food sprays, nectar and pollen sources, alternative
prey) (Figure 9).
 Thirteen of the reviews recommended that CBC research should make more use of
particular methodologies (Figure 8). Modelling was singled out by five of the reviews as a
priority and long term studies by three (Figure 9).

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 Nine reviews recommended more research on non-arthropod natural enemies, seven of
these advocating that entomopathogens were worthy of more study and two advocating
entomopathogenic nematodes (Figure 8, Figure 9).
 Only eight reviews specifically mentioned IPM or precision farming in their
recommendations for further research (Figure 8). Five of those that did so recommended
that more research effort should be applied to the integration of CBC into IPM (Figure 9).
 The socio-economic drivers of the uptake of CBC by farmers were mentioned as a priority
for further research by only three of these science-based reviews (Figure 8).
 Spatial and temporal factors were considered important in relation to many of the research
categories discussed in this section (above). Further study of the effect of spatial temporal
factors on the potential effectiveness of CBC was recommended by 43 and 13 of the 90
review papers, respectively.
Figure 8: Categories of science underpinning CBC that were identified by review papers to need
further research.

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3.5.4. Other challenges to the implementation of CBC discussed in the
literature
 Challenges to CBC implementation identified in the review papers were allocated to five
categories and 14 sub-categories (Figure 10, Figure 11; see Appendix 9 for more complete
definitions of the sub-categories).
 These largely non-scientific challenges to CBC implementation were mentioned less
frequently by the 90 scientific review papers than were scientific gaps in the knowledge-
base (Figure 8, Figure 10).
 A lack of interdisciplinary research, e.g. the division of practitioners into different ecological,
agronomic and socio-economic disciplines and sub-disciplines, was seen to hamper
scientific advancement in support of CBC by five of the reviews (Figure 11).
 High research and development costs were seen to be a challenge to the implementation of
CBC by 7 reviews (Figure 10), particularly in relation to the cost of large landscape-scale or
long term studies, the cost of semiochemical registration and the difficulty of creating a
saleable commodity to provide a return on research investment (Figure 11).
 Eight reviews cited knowledge transfer as a challenge to CBC implementation (Figure 10).
They advocated the development of improved knowledge transfer methods and highlighted
the complexity introduced by the effect of local ecological variation on CBC success (Figure
11). Two reviews regarded a shortage of taxonomic expertise and training materials for
natural enemy identification as a problem.
 Socio-economic factors were considered to be potential impediments to CBC
implementation by 7 reviews. The perceived risk of implementing CBC (lack of consistent
evidence for success), its perceived complexity in comparison to conventional chemical-
based control and the transitional costs of establishing CBC were cited. Cultural
conservatism was considered a potential problem by two reviews.
 Six reviews discussed potential challenges attending policymakers in the development and
implementation of agri-environment policy to support establishment of CBC (Figure 10). In
particular they discussed the difficulty of designing policy to promote large-scale landscape
changes that would be implemented through individual farmers who make their living at
smaller spatial scales (Figure 11). One review mentioned that the multiple functions of agri-
environment schemes led them to be complex and another highlighted the challenge facing
policymakers of increasing both crop production and biodiversity.
Figure 10: Categories of challenge to the implementation of CBC that were identified by review
papers.

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Figure 11: Categories and sub-categories of challenge to the implementation of CBC that were
identified by review papers.
3.6.Results: Management of plant pathogens
For the management of plant diseases conservation biological control is achieved either by
preserving the fraction of the micro-flora with antagonistic or competitive effects against the plant
pathogens or by active measures aiming to foster their survival or their development at the
expense of the plant pathogens (van Driesche & Bellows, 1996).
3.6.1. Biological control of air-borne pathogens
Biological control strategies against air-borne pathogens are seldom conceived as anything but
deployment of inoculative/inundative releases (Hajek, 2004) and in the scientific literature,
Conservation Biological Control is very rarely associated with the management of air-borne
diseases, whether in terms of field use or even merely as a research topic.
Nevertheless, naturally present epiphytic micro-organisms with competitive or antagonistic effects
against air-borne plant pathogens may play a greater than suspected role in protecting plant
surfaces and an analysis of what little information is available suggests that the potential of CBC
approaches in the canopy should not be overlooked. For example, Dik and VanPelt (1992) have
concluded, from field studies on the control of Septoria leaf blotch, that naturally occurring
saprophytes on the surface of wheat leaves should be protected (by eliminating harmful chemicals)

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to enhance protection. In the last 20 years, much progress has been made in the comprehension
of microbial communities in the phyllosphere (see recent review by Whipps et al. 2008) and ways
to manipulate their composition have been explored (Nix-Stohr et al. 2008) and could offer a
potential for biological control.
Interestingly, conservation biological control strategies set in place for the control of arthropod
pests may also have beneficial effects against air-borne diseases (Gurr et al. 2007). In New
Zealand, manipulating the floor of the vineyards has provided much success and attracted interest
from industry for the management of Epiphyas postvittana. In a similar way, application of mulch
was used successfully to enhance soil microbial activity and degradation of vine debris, resulting in
significant reduction in a major source of primary airborne inoculum of Botrytis cinerea (Jacometti
et al., 2007).
Conservation Biological Control represents a virtually untapped resource for the management of
air-borne diseases and much work will be necessary to fully explore this promising area. Possible
avenues for future research include:
- manipulate the resident phylloplane microflora to foster the establishment of
components with the highest effects against air-borne plant pathogens. Microbial
ecology studies will be required to identify those beneficial components of the
phyllosphere microflora and the key factors that regulate their populations. Models
describing their interactions will be needed to allow the screening of large numbers of
scenarii and evaluate the usefulness of various manipulative approaches.
- breed plant varieties able to harbour a larger fraction of the natural phylloplane
microflora with antagonistic or competitive effect against selected major airborne plant
pathogens.
3.6.2. Biological control of soil-borne pathogens
3.6.2.1. Context and definitions.
Biological control of soil-borne diseases was proposed more than forty years ago. Indeed, a
symposium held in Berkley in 1965 was entitled: “Ecology of soil-borne plant pathogens; prelude to
biological control” (Baker & Snyder, 1965). The two main approaches to biological control of soil-
borne pathogens were already proposed: enhancement of the naturally occurring populations of
antagonists and introduction of a selected biological control agent.
These two strategies can be respectively compared to “conservation biological control” and
“inoculation biological control” as defined by Eilenberg (2006). “Inoculation biological control” is
“the intentional release of a living organism as a biological control agent with the expectation that it
will multiply and control the pest for an extended period, but not permanently”. On the other hand,
“Conservation biological control” is the modification of the environment or existing practices to
protect and enhance specific enemies or other organisms to reduce the effects of pests”.
In the case of soil-borne diseases, the pathogens are always included in the soil matrix; this lead to
the concepts of “soil inoculum potential” and “soil suppressiveness to diseases”. The soil inoculum
potential is the soil-dependent capacity of pathogens to incite disease, and it results from the
interactions between inoculum density and soil suppressiveness. Soil suppressiveness
corresponds to the global effects of the soil microbiota interacting with the pathogens. Two main
mechanisms are responsible for soil suppressiveness: general suppression, which is correlated
with the activity of the total microbial biomass at critical times for the pathogen, and specific
suppression which is due to the activity of specific micro-organisms that are antagonistic to the
pathogen (Cook & Baker, 1983). Soil suppressiveness being essentially biological, it is possible to
increase soil suppressiveness by cultural practices that influence different aspects of soil biology.
Managing cultural practices in order to increase soil suppressiveness to diseases corresponds to
“conservation biological control” as defined above.

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3.6.2.2. Cultural practices used for conservation biological control of soil-
borne diseases.
Crop rotation
As a rule, continuous cropping of the same plant species will lead to an increase in incidence of
soil-borne diseases, while rotation with non-hosts should lead to a decrease in incidence. There
are few exceptions to this general law that mono-cropping will increase disease. The best known
example is that of take-all decline: after increasing during a few years (4-5) disease severity will
decrease, to such a level that the yield will not be affected by the disease (Hornby, 1998). This is a
clear example of continuous cropping altering disease suppressiveness of soil through its effects
on specific components of the soil microflora. On the contrary, most of the diseases induced by soil
borne plant pathogens could be controlled by an appropriate crop rotation sequence. The main
effect of crop rotation is to allow time for decrease, through natural mortality, of inoculum of
pathogens that are poor saprophytic competitors. Clean fallows have the same mechanism.
However, since mortality of pathogen propagules in soil is frequently due to the effects of other
organisms, the stimulation of microbial activity by the growing rotational crop should make rotation
more effective than fallowing. Crop rotation increases the diversity of plants within an agricultural
system, which may have effects on the diversity of soil biota (Lupwayi et al., 1998).
Tillage
Soil disturbance by tillage has been shown to have a variety of effects on diseases. Root rots of
many crop plants caused by R. solani are generally less severe after tillage than with direct drilling
(Roget et al., 1985). On the other hand, common root rot of wheat caused by Cochliobolus sativus
may be more severe in tilled soils (Mathieson et al., 1990). These well-characterized effects of
tillage on disease seem to act directly on the pathogen, with no evidence yet of effects on other
components of the soil biota. However, tillage is expected to have some influence on soil
suppressiveness because it does alter the activity and diversity of soil microflora. Typically, tillage
reduces bacterial biomass and diversity in soil, possibly through its effects on soil aggregation
(Lupwayi et al., 2001). Reduced tillage systems should therefore have more diverse and active
microflora, and greater general suppression of diseases.
Residue management
Residue management can have conflicting effects on disease. Retaining residues increases the
inoculum potential of pathogens that survive in the residue. On the contrary, residue retention can
boost the levels of general suppression in soils. Indeed, general suppression has been linked to
high levels of microbial activity, which depend on high levels of OM input into soils. Moreover,
residue retention may favour specific antagonists increasing the level of specific suppression. For
example, populations of cellulolytic organisms tend to be higher in soils where crop residues are
retained and high cellulolytic activity has been correlated with suppression of disease such as
Fusarium seedling blight in barley ( Papavizas, 1985).
Solarisation
Solarisation or solar heating is a method that uses the solar energy to enhance the soil
temperature and reach levels at which many plant pathogens will be killed or sufficiently weakened
to obtain significant control of the diseases. Solarisation does not destroy all the soil micro-
organisms, but modifies the microbial balance in favour of the beneficial micro-organisms. Efficacy
of soil solarisation is not only due to a decrease of the pathogenic populations but also to an
increase of the density and activity of populations of micro-organisms antagonistic to the
pathogens. Soil solarisation is really a conservation biological control practice. It possesses a very
large spectrum of activity; it controls fungi, nematodes, bacteria, weeds, arthropod pests and some
unidentified agents (Katan, 1996).
Biofumigation or biodisinfection
Biological soil disinfection is based on plastic tarping of the soil after incorporation of fresh organic
matter. The mechanisms involved are not totally understood. Fermentation of organic matters
results in the production of toxic metabolites and in anaerobic conditions which both contribute to
the inactivation or destruction of the pathogenic fungi. Based on the dominant type of mechanisms

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involved, it was proposed to make the distinction between (i) biofumigation which corresponds to
the use of specific plant species containing identified toxic molecules, and (ii) biodisinfection which
refers to the use of high quantities of organic matter which results in anaerobic conditions mainly
responsible for the destruction of the pathogens (Lamers et al., 2004).
Compost amendments
Addition of organic amendments such as animal manures and industrial by-products is the best-
documented strategy for increasing disease suppression in soils. Manures and other amendments
tend to increase microbial biomass and biological activity in soil, and thus to enhance general
suppression. Composting organic matter is an interesting process enabling to transform wastes
from different origin in composts which are beneficial for soil health. This is a biological process
characterized by a heat peak which destroys the thermo-susceptible micro-organisms, resulting in
compost free from most plant pathogens Most of these composts possess the capacity to increase
soil suppressiveness to diseases However, there is no universal rule; the level of disease control
obtained depends on many factors such as the chemical properties of the parent materials, the
composting process, the types of micro-organisms which colonized the compost after the heat-
peak and obviously the type of plant pathogens to be controlled (Termorshuizen et al., 2006).
3.6.3. Conclusions
These management practices that contribute to control soil-borne plant pathogens are not
exclusive from other biological control methods. On the contrary they should be used in association
with other biological methods such as the use of specific biological control agents. However it is
our opinion that these conservation biological control methods have been neglected. Obviously
there are much more difficult to apply than other control methods and their efficacy depends on
many factors which are not easy to control. But the sustainable approach requires that all the
available methods should be used in association in order to drastically reduce the use of chemical
pesticides.
3.7.Results: Management of weeds
Biological control of weeds has to date largely focussed on the release of agents as ‘bioherbicides’
in augmentative release, inundative release or classical biological control programmes (Rao,
2000). The development of conservation strategies for weed biological control agents has been
directed at these released agents (fungal pathogens, rhizobacteria, insects and nematodes).
Considerably less research has been directed towards CBC strategies that optimise the impact of
naturally-occurring populations of weed natural enemies than has been done for invertebrate
pests. There is a view that, although naturally-occurring levels of biological control are typically
high (particularly through seed predation), the control exerted is not easily manipulated and is thus
of limited value as a management tool (Norris, 2007). Nevertheless, there are good examples of
native agents that control weeds that might be successfully exploited should management
strategies be established to conserve them (Newman et al., 1998).
Factors influencing the conservation of naturally-occurring biological control agents in CBC
strategies are likely to be similar to those affecting released agents. These include the timing and
nature of disturbances such as tillage, grazing, mowing, harvesting and pesticide applications, the
choice of crop rotation and the provision of habitats and refugia (Newman et al., 1998, Rao, 2000).
The biological control agents that have received most attention in weed CBC research are
deleterious rhizobacteria, and granivorous carabids, ants and small rodents. The techniques that
appear to have most potential for weed CBC are the management of crop residues by
conservation tillage and by manipulation of crop rotations, and the management of habitats (refugia
and resources) for invertebrates. Both deleterious rhizobacteria and carabids can benefit from the
accumulation of crop residues in the soil or at its surface.

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Deleterious rhizobacteria in CBC strategies for weeds
Amongst the many cultural control methods used for weed management are the use of mulches
and residues with allelopathic properties (Shennan, 2008). There is good evidence that such soil
management practices lead to accumulation of organic matter and to increased soil enzyme
activity that can be associated with suppression of both plant diseases and weeds (Kremer and Li,
2003). In soils thus managed, the diversity of microbial populations is increased and the potential
to develop weed-inhibiting bacterial communities is enhanced (Kremer and Li, 2003). Among such
bacterial communities are deleterious rhizobacteria that can be isolated from rhizosphere soils and
can inhibit weed growth (Ibekwe and Kennedy, 1999, Kremer and Kennedy, 1996).
It has been proposed that crop residue management could be specifically designed for CBC
strategies to encourage the strains of naturally-occurring deleterious rhizobacteria that are harmful
to weeds (Kremer and Li, 2003, Rao, 2000). Moreover, it is possible that crop rotations could be
redesigned to optimise the development of specific strains of rhizobacteria for weed suppression
because particular rhizobacteria can be associated with the roots of particular plant species (e.g.
maize; Turco et al., 1990, Rao, 2000).
Insects in CBC strategies for weeds
There is good evidence that native populations of phytophagous insects can be managed to exert
substantial control on weed species. For example, snakeweeds and locoweeds, native weeds of
rangeland in the south-western, USA can be substantially controlled by native grasshoppers and
root-boring beetles if prescribed burns and insecticide applications are timed to avoid vulnerable
points in their life-cycle (Newman et al., 1998). Furthermore, both experimental and modelling
studies show that predation of weed seed by invertebrates has the potential to reduce the weed
seed-bank (Menalled et al., 2005).
A significant number of species of carabid beetle are predominantly or in part phytophagous and
some of these have a granivorous habit (especially in the genera Amara, Harpalus, Ophonus and
Zabrus) that gives them particular potential in CBC strategies for weed control. In warm temperate
areas such as the Mediterranean region, ants are more significant seed predators and in some
crops (e.g. winter cereals) they are the dominant seed predators (Baraibar et al., 2009).
Intensive high-input agriculture has been accompanied by a decline in populations of carabids in
farmland (Kromp, 1999) and so measures to conserve and promote them are needed if their
influence on weed control is to be maximised. Ploughing reduces the survival of many carabid
species (Holland, 2004). By contrast, populations of many invertebrates are enhanced in minimum
tillage or conservation tillage regimes where soil disturbance is reduced and a richer habitat is
provided by the presence of crop residues and greater weed diversity (Holland, 2004, Kromp,
1999). Weed seed predation by carabids, ants and mice is higher in no-tillage than in conventional
systems (Brust and House, 1988, Baraibar et al., 2009) and it has been suggested that a
significant proportion of the influence of crop residues in suppressing broadleaf weeds in low-input
no-tillage systems is due to seed predation by carabids (Brust, 1994). These positive benefits of
no-tillage need to be balanced against the effectiveness of ploughing in suppressing weed
populations by burying weed seeds.
Habitat manipulation is believed to be an important means of promoting natural enemies of insect
pests (see Section 5: Results: Management of invertebrate pests) and may have a similar potential
for increasing weed seed predation (Landis et al., 2005). There is evidence that weed seed
predation can be limited by a shortage of suitable habitats and refugia for herbivores (Diaz, 1994)
and that weed seed predation (both pre-dispersal and post-dispersal) is greater in complex
landscapes than in simple ones (Menalled et al., 2000, Steffan-Dewenter et al., 2001). Crop and
landscape diversification by intercropping and the provision of enhanced boundary habitats, such
as sown weed strips, beetle banks and conservation headlands, generally enhance carabid
diversity and promote the populations of some species in farmland (Kromp, 1999). However, the
impact of such measures will depend on the extent of the extent of ingress of margin beetle
communities into the field centre (Collins et al., 2002) and the degree of synchrony of beetle

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activity with weed seed production. Management of boundary habitats and of cropped areas also
infuences the activitiy of rodent seed predators. For example, cover crops may be valuable for
maintaining activity of rodents in fields (Westerman et al., 2006).
Research gaps that represent barriers to the implementation of CBC strategies for weeds
The development of CBC strategies for weeds is in its infancy and considerable research effort is
needed if strategies are to be developed and tested and if risks and benefits are to be explored to
the satisfaction of both researchers and growers. Research should focus both on the ecology of
relationships between weeds and their natural enemies and on assessment of the impact of CBC.
Quantification of the effectiveness, reliability and cost of CBC strategies for weeds under realistic
field conditions is particularly important if it is to be adopted into farm practice.
The following research needs were particularly highlighted in the literature reviewed:
 Studies of the influence of soil aggregate characteristics on soil enzyme activity to elucidate
the relationship between the soil type and the microbial community supported, including
deleterious rhizobacteria (Kremer and Li, 2003).
 In-depth research on the ecology of relationships between deleterious rhizobacteria and
plants and on the mechanisms of action against weeds, including characterization of
phytotoxins (Kremer and Kennedy, 1996).
 Design of crop rotations to optimise the development of specific strains of deleterious
rhizobacteria for weed suppression (Rao, 2000).
 Integration of conservation strategies for natural enemies of weeds into weed management
(Newman et al., 1998).
 Studies of pesticide targeting to conserve native biological control agents (Newman et al.,
1998).
 A comprehensive study of the ecology of predation of weed seed by invertebrates and
vertebrates and its impact on weed populations (Menalled et al., 2005).
 Research is needed on manipulation of the soil environment to encourage predators and
pathogens of weed seed, e.g. by conservation tillage systems (Derksen et al., 1996).
 Research on habitat protection and plant community management (e.g. in field margins) to
conserve critical habitats or refugia for weed seed predators (Newman et al., 1998).
 Demonstration of the extent that landscape diversification benefits carabid populations
within cropped land (Kromp, 1999) and assessment of the impact on weed control.
 Studies to quantify the costs and benefits of promoting carabids and to assess their
reliability in weed control (Kromp, 1999).
 Rigorous evaluation of the effectiveness of weed biological control projects and the reasons
for success or failure (Newman et al., 1998).
3.8.References cited in Chapter 3
(For a list of reviews used in the meta-review of CBC of invertebrate pests, see Appendix 2)
Baker KF and Snyder WC. 1965. Ecology of Soil-Borne Plant Pathogens. Prelude to Biological
control. University of California Press Berkley. 571pp.
Baraibar, B., Westerman, P.R., Recasens, J., 2009. Effects of tillage and irrigation in cereal fields
on weed seed removal by seed predators. Journal of Applied Ecology 46, 380-387.
Brust, G.E., House, G.J., 1988. Weed seed destruction by arthropods and rodents in low-input
soybean agroecosystems. American Journal of Alternative Agriculture 3, 19-25.
Brust, G.E., 1994. Seed-predators reduce broadleaf weed growth and competitive ability. Agric.
Ecosyst. Environ. 48, 27-34.
Collins, K.L., Boatman, N.D., Wilcox, A., Holland, J.M., Chaney, K., 2002. Influence of beetle banks
on cereal aphid predation in winter wheat. Agric. Ecosyst. Environ. 93, 337-350.
Cook R, Baker KF. 1983. The nature and practice of biological control of plant pathogens. St Paul,
MN: The American Phytopathological Society. 539pp.

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4. Classical and augmentative biocontrol: critical status analysis
for selected crops
Contributors:
Nicot Philippe, INRA, Plant Pathologist, (general coordination & editing; biocontrol of diseases;
measures for improvement)
Alabouvette Claude, INRA, Plant Pathologist (biocontrol of Fusarium; regulatory issues)
Bardin Marc, Plant Pathologist (biocontrol of Botrytis, rusts)
Blum Bernard, IBMA (economic survey, regulatory issues)
Giorgini Massimo, CNR, Entomologist (classical and augmentative/inundative biocontrol)
Heilig Ulf, IBMA (commercial products, regulatory issues)
Köhl Jürgen, WUR, Plant Pathologist (biocontrol of Venturia; Ulocladium)
Malausa Jean Claude, INRA, Entomologist (classical biocontrol and parasitoids)
Ris Nicolas, INRA, Entomologist (classical biocontrol and parasitoids)
Ruocco Michelina, CNR, Plant Pathologist (efficacy at field level; Trichoderma)
4.1.Potential of biocontrol based on published research
4.1.1. Review of the scientific literature on biocontrol against the main plant
pathogens of selected crops
4.1.1.1. Evolution of the scientific literature 1973-2008
The scientific literature published in the last 35 years comprises a wealth of studies on biological
control against diseases and pests of agricultural crops. A survey of the CAB Abstracts® database
shows a steady increase in the yearly number of these publications from 20 in 1973 to over 700
per year since 2004 (Figure 12).
0
100
200
300
400
500
600
700
800
900
1970 1975 1980 1985 1990 1995 2000 2005 2010
Publication year
Numberofpublicationsperyear
Figure 12: Evolution of the yearly number of publications dedicated to biological control of plant
diseases based on a survey of the CAB Abstracts® database.
This survey was further refined by entering keywords describing some of the major plant
pathogens/diseases of cultivated crops in Europe, alone or cross-referenced with keywords

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indicating biocontrol. Among studies published in the period between 1973 and 2008 on these
plant pathogens and pests, the percentage dedicated to biological control was substantial, but
unequally distributed (Table 11). It was notably higher for studies on soil-borne (9.5% ± 1.6% as
average ± standard error) than for those on air-borne diseases (2.8% ± 0.7%).
Table 11: Scientific papers published between 1973 and 2008 on biological control against
major plant diseases (from CAB Abstracts® database).
Disease or plant pathogen
Total number
of references
References on biological control
%
Soil-borne:
Fusarium 34 818 1 925 5.5
Rhizoctonia 10 744 1 278 11.9
Verticillium 7 585 592 7.8
Pythium 5 772 821 14.2
Sclerotinia 5 545 456 8.2
Air-borne:
rusts 29 505 360 1.2
powdery mildews 18 026 251 1.4
Alternaria 12 766 415 3.3
anthracnose 12 390 351 2.8
Botrytis 9 295 705 7.5
downy mildews 8 456 80 1.0
Phytophthora infestans 5 303 61 1.1
Monilia rot 1 861 81 4.3
Venturia 3 870 104 2.7
4.1.1.2. Inventory of potential biocontrol agents (microbials, botanicals,
other natural compounds)
The scientific literature described above was further examined to identify biocontrol compounds
and microbial species reported to have a successful effect. Due to the great abundance of
references, it was not possible to examine the complete body of literature. The study was thus
focused on several key diseases selected for their general importance on cultivated crops, and in
particular on those crops studied in ENDURE case studies.
 Methodology:
Three steps were followed.
The first step consisted in collecting the appropriate literature references for the selected key
diseases/plant pathogens to be targeted by the study. The references were extracted from the
CAB Abstracts® database and downloaded to separate files using version X1 of EndNote (one file
for each target group). The files were then distributed among the contributors of this task for
detailed analysis.
In the second step, every reference was examined and we recorded for each:
- the types of biocontrol agents (Microbial, Botanical or Other compounds) under study and
their Latin name (for living organisms and plant extracts) or chemical name
- the Latin name of the specifically targeted pathogens,
- the crop species (unless tests were carried out exclusively in vitro),

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- the outcome of efficacy tests.
Two types of efficacy tests were distinguished: Controlled environment tests (including tests on
plants and in vitro tests), and field trials. The outcome of a test was rated (+) if significant effect
was reported, (0) if no significant efficacy was shown and (-) if the biocontrol agent stimulated
disease development.
To allow for the analysis of a large number of references, the abstracts were examined for the
presence of the relevant data. The complete publications were acquired and examined only when
the abstracts were not sufficiently precise.
The data were collected in separate tables for each type of key target pest. For each table, they
were sorted (in decreasing order of priority) according to the type and name of the biocontrol
agents, the specifically targeted pest, and the outcome of efficacy tests.
In the third step, synthetic summary tables were constructed to quantify the number of different
biocontrol compounds and microbial species and strains reported to have successful effect against
each type of key pathogen/disease or pest target.
 Results
A total number of 1791 references were examined for key airborne diseases including powdery
mildews, rusts, downy mildews (+ late blight of Potato/Tomato) and Botrytis and Monilia rots,
together with soilborne diseases caused by Fusarium oxysporum (Table 12). Based on the
examination of these references, successful effect in controlled conditions was achieved for all
targets under study with a variety of species and compounds (Appendices 10-15, Table 13).
Table 12: Numbers of references on biocontrol examined per group of disease/plant pathogen.
Target disease / plant
pathogen
Relevance to
ENDURE Case
Studies
Number of
references
examined
Period of
publication
examined
Contributor
Botrytis
OR, FV, GR*
(postharvest)
880 1998-2008 INRA
Powdery mildews all 166 1998-2008 CNR
Rusts AC, FV, OR 154 1973-2008 INRA
Downy mildews +
Phytophthora infestans
FV, GR, PO, TO 349 1973-2008 INRA
Monilinia rot OR 194 1973-2008 INRA
Fusarium oxysporum FV, TO 48 2007-2009 INRA
*AC: Arable Crops; FV: Field Vegetables; GR: Grapes; OR: orchard; PO: Potato; TO: Tomato
Concerning airborne diseases and pathogens, the largest number of reported successes was
achieved with microbials, but there is a growing body of literature on plant and microbial extracts,
as well as other types of substances (Table 13). On average, reports of success were far more
numerous for experiments in controlled conditions (in vitro or in planta) than for field trials.
Very contrasted situations were also observed depending on the type of target
disease/pathogen, with rare reports on the biocontrol of rusts and mildews compared to Botrytis,
despite the fact that the literature was examined over a 35 year period for the former diseases and
only over the last 10 years for the latter.
In total in this review, 157 species of micro-organisms have been reported for significant
biocontrol activity. They belong to 36 genera of fungi or oomycetes, 13 of yeasts and 25 of
bacteria. Among them, 29 species of fungi/oomycetes and 18 bacteria were reported as successful
in the field against at least one of the five key airborne diseases included in this review (Table 14).

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P. agglomerans
fruits, grapes,
legumes, strawberry
legumes
apple, apricot,
blueberry,
cherry, peach,
plum
Pseudomonas spp.
flowers, fruits, grapes,
field vegetables
others
potato, tomato,
field vegetables
apricot
P. aeruginosa protected vegetables
P. aureofasciens cereals cherry
P. cepacia strawberry peach
P. chlororaphis strawberry cherry
P. corrugata peach
P. fluorescens
fruits, grapes,
legumes, strawberry,
protected vegetables,
others
cereals, ,
protected
vegetables
others
legumes
grapes, potato,
tomato, others
blueberry,
cherry
P. putida
flowers, legumes,
protected vegetables,
others
cereals
P. syringae
fruits, strawberry, field
vegetables
grapes apple, peach
P. reactans strawberry
P. viridiflava fruits
Rhanella spp. potato
R aquatilis fruits
Serratia spp. potato
S. marcescens flowers
S. plymuthica protected vegetables
Stenotrophomonas
maltophilia
legumes
Streptomyces spp. tomato
S. albaduncus legumes
S. ahygroscopicus protected vegetables
S. exfoliatus legumes
S. griseoplanus legumes
S. griseoviridis
protected vegetables,
others
S. lydicus protected vegetables
S. violaceus legumes
Virgibacillus marismortui strawberry
Xenorhabdus bovienii potato
X. nematophilus
protected
vegetables
One striking aspect of this inventory is that although the five target diseases / pathogens
included in our review are airborne and affect mostly the plant canopy, the vast majority of cited
biocontrol microorganisms are soil microorganisms. The scarcity of biocontrol agents originating
from the phyllosphere could be due to actual lack of effectiveness, or it could be the result of a bias
by research groups in favour of soil microbes when they gather candidate microorganisms to be
screened for biocontrol activity. This question would merit further analysis as it may help to devise
improved screening strategies. As "negative" results (the lack of effectiveness of tested
microorganisms, for example) are seldom published, the completion of such an analysis would in
turn necessitate direct information from research groups who have been implicated in screening for
biocontrol agents, or the development of a specific screening experiment comparing equal
numbers of phyllosphere and of soil microbial candidates.
Another striking aspect is that most of the beneficial micro-organisms inventoried in this study
(49 fungi/oomycetes, 28 yeasts and 41 bacteria) are cited only for biocontrol of one of the five
types of airborne diseases included in the survey (Figure 13). However, several species clearly
stand out with a wide range of effectiveness, as they were successfully used against all five types
of target diseases on a variety of crops. This includes the fungi Trichoderma harzianum and

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Trichoderma viride (2 of 12 species of Trichoderma reported as biocontrol-effective in the reviewed
literature) and the bacteria Bacillus subtilis and Pseudomonas fluorescens.
0
5
10
15
20
25
30
35
40
45
50
1 2 3 4 5
Number of controlled target diseases / pathogens
Numberofspecies
fungi / oomycetes
yeasts
bacteria
Figure 13: Range of efficacy of 157 microbial biocontrol agents against five main types of airborne
diseases. Detailed data are presented in Table 14.
Concerning Fusarium oxysporum. Data base interrogation was done on October 6, 2009.
With the key words “Fusarium oxysporum AND biological control” the numbers of references were
as follows:
1973-2009: 2266 references
1999-2009: 1426 references
2004-2009: 899 references
2007-2009: 502 references
Using these key words we did not select only papers regarding biological control of diseases
induced by F. oxysporum but also all the paper dealing with the use of strains of F. oxysporum to
control diseases and weeds. There are quite many papers dealing with the use of different strains
of F. oxysporum to control Broom rape (orobanche) and also the use of F. oxysporum f. sp.
erythroxyli to eradicate coca crops.
We decided to limit our review to the two last years and to concentrate on references for which
full text was available on line.
Finally we reviewed 48 papers. All these papers were dealing with the selection and
development of micro-biological control agents; only two were considering others methods. One
was addressing the use of chemical elicitors to induce resistance in the plant; the other was aiming
at identifying the beneficial influence of non-host plant species either used in rotation or in co-
culture.
Based on this very limited number of papers we identified;
The formae speciales of F. oxysporum the most frequently studied:
F.o. f. sp. lycopersici is the model pathogen in 17 papers,
f. spp. melonis, ciceris, cubense, niveum and cucumerinum are used in 2, 3 or 4 studies.
The antagonists studied:
Bacillus spp and Paenibacillus are considered in 16 papers, B. subtilis being the most frequently
used

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Trichoderma spp. are considered in 14 papers
Fluorescent Pseudomonas spp in 7 papers
Actinomycetes in 5 papers
Non pathogenic strains of F. oxysporum in 5 papers
Mycorrhizal fungi in 3 papers
Penicillium in 2 publications
Most of the publications (28) are reporting in vitro studies. Among them a few are studying in
vitro, or in planta the mechanisms of action of the antagonists, the others just relate screening
studies using plate confrontation between the antagonists and the target pathogens. In most of
these papers (22) the in vitro screening is followed by pots or greenhouses experiments aiming at
demonstrating the capacity of the antagonist to reduce disease severity or disease incidence after
artificial inoculation of the pathogen. Finally only 9 publications report results of field experiments.
Most of these papers conclude on the promising potential of the selected strains of antagonists
able to decrease disease incidence or severity by 60 to 90 %.
In contrast to these optimistic results, there is still no preparation on the market targeting control
of Fusarium wilts. The strains of Trichoderma on the market sometimes claim efficacy against
Fusarium oxysporum, but they are mostly used to control damping-off and root-rot, not wilt.
Similarly the strains of Bacillus subtilis already on the market are not targeting Fusarium wilts.
In the eighties, a strain of Pseudomonas fluorescens was developed in the Netherlands to
control Fusarium wilt of radish, but it is no more on the market.
Only a very few teams are regularly publishing on biological control of Fusarium wilts. They are
interested in all the aspects from the modes of action of the antagonists, the plant-fungal
interactions, the fate of the antagonists in the environment and the processes of production and
formulation of the biological control product.
The most promising results concern the use of non pathogenic strains of F. oxysporum and of a
strain of Penicillium oxalicum.
Generally speaking, this limited literature review shows that most of the lab studies are not
followed by field studies. There is a need for implementation of biological control in the fields.
 Identified knowledge gaps
Several types of knowledge gaps have been identified in this review. They include:
- the near absence of information on biocontrol against diseases of certain important European
crops such as winter arable crops.
- the scarcity of reports on biocontrol against several diseases of major economic importance
on numerous crops, such as those caused by obligate plant pathogens (rusts, powdery
mildews, downy mildews)
- the still limited (but increasing) body of detailed knowledge on specific mechanisms of action
and their genetic determinism. The little knowledge available at the molecular level is
concentrated on few model biocontrol agents such as Trichoderma and Pseudomonas.
- the still very limited information on secondary metabolites produced by microbial biocontrol
agents
- the lack of understanding for generally low field efficacy of resistance-inducing compounds
- the lack of knowledge on variability in the susceptibility of plants pathogens to the action of
BCAs and on possible consequences for field efficacy and its durability.

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4.1.2. Review of the scientific literature on beneficials for classical and
augmentative/inundative biocontrol against insect pests
4.1.2.1. Bibliographic survey on augmentative biological control against arthropod
pests in selected crops
We carried out a preliminary bibliographic survey to quantify the literature on augmentative
biological control of pests published from 1973 to 2008. The survey was restricted to crops relevant
to case studies of ENDURE. They included grapevine; orchards: apple and pear; arable crops:
corn and wheat; field vegetables: carrot and onion. Augmentative biological control (Van Driesche
& Bellows, 1996) comprises of inoculative augmentation (control being provided by the offspring of
released organisms) and inundative augmentation (control expected to be performed by the
organisms released, with little or no contribution by their offspring).
Our bibliographic survey was conducted by the CAB Abstracts database by entering the
name of each crop and one key word selected from the following list in order to retrieve the
maximum number of references. For each selected crop, the key words used for the bibliographic
survey were: a) augmentative biological control; b) augmentation biological control; c) inoculative
biological control; d) inundative biological control. The survey with these key words produced a
very low number of results all of which were examined. For this reason we added two key words
that were more general: e) insects biological control; f) mites biological control. For the searching
criteria a to d, total records will be examined. In this case, given the extremely high number of
records, only references within the period 1998-2008 were examined to select only the publications
concerning the augmentative biological control. The results of this survey are reported in Appendix
16.
The analytical review of the scientific literature on augmentative biological control has been
done only for grapevine.
4.1.2.2. Status of researches on augmentation of natural enemies to control
arthropod pests in grapevine
The references extracted from the CAB Abstracts database, following the criteria described
in the previous paragraph, were examined to identify those concerning the use of natural enemies
in augmentation biological control in grapevine. The abstracts of 607 references were examined
and only 70 papers reported data on application and efficiency of augmentative biocontrol (Table
15).
Table 15: References extracted from the CAB Abstracts database and examined for reviewing
augmentation biological control in grapevine.
Key words Total records
(1973-2008)
1998-2008
Augmentative biological control 7 6
Augmentation biological control 10 6
Inoculative biological control 4 1
Inundative biological control 7 3
Insects biological control 373
Mites biological control 190
Total references examined 28 579
Total references showing data on
augmentative biocontrol
70
The survey includes records for grapevine, grape and vineyard.

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 Results
Very few papers (62) on augmentative biocontrol in grapevine have been published during
the period 1998-2008, with an average of 5.6 publications per year. Most references (93.5%)
showed data on biological control of insects and only 4 papers on the biological control of mites
were published (Figure 14).
The data extracted from the abstracts of the selected references were collected analytically
in separate tables for each group of biocontrol agents (Appendix 17): references were sorted
chronologically (starting from the eldest). For each species of biocontrol agent, target species of
pest, Country, type of augmentation (inundative, inoculative), type of test (laboratory, field), efficacy
of biocontrol, additional information and results were reported.
Data reported in Appendix 17 were summarized in Table 16, Table 17, Table 18, Figure 15
and Figure 16. A list of the biocontrol agents used in augmentative biological control in grapevine is
reported in Table 16 and Figure 15. A list of groups and species of the targeted pests and the
antagonists used for their control is reported in Table 17 and Figure 16; the efficacy of biocontrol
agents is reported in Table 18.
The group of pests on which the highest number of augmentative biocontrol researches has
been carried out is Lepidoptera (60% of total references) with the family Tortricidae representing
the main target (55%) (Figure 16) including the grape berry moths key pests Lobesia botrana and
Eupecilia ambiguella (Table 17). Bacillus thuringiensis has resulted the most frequently used
biocontrol agent against Lepidoptera by achieving an effective control of different targets in
different geographic areas (Table 17, Table 18, Appendix 17.7). We sorted 28 references (39% of
the total citations) dealing with the use of B. thuringiensis of which 23 references were referred to
the control of L. botrana. The augmentation of egg parasitoids of the genus Trichogramma
(Hymenoptera: Trichogrammatidae) resulted the alternative strategy to B. thuringiensis to control
Lepidoptera Tortricidae (13 references, 16% of total citations) (Table 17, Table 18). Field
evaluations indicated T. evanescens as a promising biocontrol agent of L. botrana (El-Wakeil et al.,
2008 in Appendix 17.1).
Fewer researches were carried out on augmentative biocontrol of other group of pests. First
in the list were mealybugs (Hemiptera: Pseudococcidae) (9 references, 13% of the total citations).
In field evaluations (4 papers) parasitoid wasps of the family Encyrtidae have resulted extremely
active and promising to be used in augmentative biocontrol of mealybugs (Appendix 17.2).
Antagonists used in augmentative biocontrol in grapevine were mainly represented by
insect pathogens (59% of the total citations), including the bacterium B. thuringiensis, fungi and
nematodes (Figure 15, Table 16). Beside the efficacy of B. thuringiensis, promising results were
obtained from researches in the control of the grape phylloxera Daktulosphaira vitifolie, a gall-
forming aphid, by soil treatments with the fungus Metarhizium anisopliae (Table 18, Appendix
17.5). Once controlled by grafting European grape cultivars onto resistant rootstocks, the grape
phylloxera has gone to resurgence in commercial vineyards worldwide and new biological control
strategy could be necessary to complement the use of resistant rootstocks and to avoid the
distribution of chemical insecticides in the soil.
Entomophagous arthropods, including parasitoid wasps and predators represented 41% of
the total citations (Figure 15, Table 16). Best results were obtained from researches on parasitoids
(18 references), namely the use of Trichogrammatidae and Encyrtidae in augmentative biocontrol
of grape moths (Tortricidae) and mealybugs (Pseudococcidae) respectively (Table 17, Table 18,
Appendix 17.1 and 17.2). Among predators, augmentation of Phytoseiidae mites has produced
some positive results in controlling spider mites and eriophyid mites on grape (Table 17, Table 18,
Appendix 17.3).

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 Brief considerations
Key pests of grapevine like L. botrana and E. ambiguella can be controlled effectively
with augmentative strategies that rely on the use of B. thuringiensis. To date, formulations of
B. thuringiensis are currently used in IPM strategies. The specificity of B. thuringiensis could
be a problem in those vineyards where other pasts can reach the status of economically
importance, if not controlled by indigenous and/or introduced natural enemies. Researches
on augmentative biocontrol should be implemented in order to develop new strategies to
solve problems related to emerging pests and alternatives to B. thuringiensis if resistant
strains should appear in target species.
4.1.3. Research and Development in classical biological control with
emphasis on the recent introduction of insect parasitoids
4.1.3.1. Scope of the review
Defined as “the intentional introduction of an exotic, usually co-evolved, biological
control agent [hereafter BCA] for permanent establishment and long-term pest control’,
classical biological control [hereafter ClBC] is a pest control strategy that has crystallized
numerous studies since more than one century and provided numerous efficient solutions for
pest control. The main advantages and risks of this strategy can be summarized as follows.
In a context of the globalisation of international trade and human mobility, an ever growing
number of exotic pests emerge locally. Such species can rapidly pullulate and jeopardize
cultural practices. This general trend can also be favoured by global climatic changes that
may allow the development of agronomic pests beyond their initial distribution area and
increase their demography. Within this context, ClBC appears often to be the first way to try
to regulate such pest populations. Moreover, when successful, ClBC appears to be very
economic insofar as financial costs are only associated with the identification, evaluation and
initial releases of exotic BCA. Contrary to other pest control strategy, the implication of
practitioners and other costs are not necessary after the establishment of the BCA. The
overall financial costs of such operations are consequently rather limited with regard to the
durability of the pest control, in particular when the local introduction of a new BCA benefits
from the previous experiences in other countries. Nevertheless, at least two kinds of risks
are usually associated with ClBC. First of all, the average success rate of ClBC varies
between 10 and 30% according to the authors for a total of more than 5000 introductions
worldwide during the last century. As consequence, such operations may also appear too
risky to be funded. Another risk is those associated with the non-target effects. Although few
cases have been reported, their echoes may have contributed to a more harmonized
approach and in some countries to more or less stringent regulations.
As consequences, classical biological programmes are at the crossroad of several
concerns:
- agronomic; insofar as each introduction of exotic BCA is obviously an hope for the
producers ;
- scientific; ClBC namely questions both ecologist and evolutionist in order to
optimize the probability of establishment while minimizing the non-target effects. Their
implication on such issues nevertheless depends on their own interest (in term of scientific
question and/or possibility or publishing);
- political; since the introduction of BCA may depend on regulation or homologation
decided at national or international levels;
- financial; since the development of ClBC is relying on various sources of funding
(agronomic partners, scientific partners, politic institutions) with various interests and
rationale (more or less short-term results, scientific excellence versus applied objectives).

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Within this context, global evaluations of ClBC programmes are necessary to better
understand the evolution of this practice and try to improve its use and efficiency. This has
been repeatedly achieved during the last years either through reviews or meta-analysis.
Based on a large (but probably not exhaustive) bibliographic survey, the present work aims
to give a complementary point of view with the willingness to portray a realistic “state of the
art” of Research and Development programmes of ClBC against arthropods. This chapter
also firstly gives a broad temporal survey of the publication and a more precise survey of the
literature for the decade [1999; 2008]. Biocontrol programmes against arthropods were then
more precisely detailed with the objectives to give qualitative cues about the main pests and
the types of related studies. Finally, a particular emphasis has been put on recent
introductions of exotic insect parasitoids.
Based on these data, we also address some more or less important subjective
recommendations based on our own opinion.
4.1.3.2. Method
A large bibliographic survey has been conducted with the CAB abstracts. Several
combinations of key-words were used with various successes. Too broad (e.g; cases for
which discussion about ClBC are marginal) or unprecise (e.g. cases for which a pest is not
precised) publications were excluded.
764 publications were found using the key-words “classical biological control” or
“classical biocontrol”. 452 papers were published during the period [1999-2008] but about
30% were not relevant with regard to the purpose of this survey and have been discarded.
329 ClBC-related publications were obtained using the more complex combinations
[“biological control” AND “exotic” AND “introduction”] but only 253 dressed precisely
questions related to classical biological control. 117 were published during the selected
temporal frame but only 81 were relevant with regard to our objectives.
47 ClBC-related publications were obtained using the more key-words association
[“biological control” AND “exotic” AND “importation”] with 17 papers for the last ten years.
Most of this literature was dedicated to the risk or regulatory aspects associated with the
importation of exotic BCA so that only 7 relevant publications with regard to our objectives.
Finally, 130 publications were found using “acclimatization” AND “biological control”
for only one relevant publication for the targeted period.
A total of 358 publications were also obtained which is probably for far from being
exhaustive. For instance, 37 new references about BCA introductions were found in addition
to the first 35 references found with the previous key-words combinations (see Table 19).
Additional bibliographic research were also realised for some taxa (see §4.1.3.3)
[Recommendation 1 (Minor - Scientists): Although the terms “classical biological
control” or “classical biocontrol” may be not as explicit as others (“introduction”,
“importation”), the generalization of their use in titles, key-words or abstracts should be
nevertheless used in order to improve the efficiency of bibliographic survey]
4.1.3.3. General trends
The temporal survey shows a quite regular increase of ClBC related publications with
a mean of about 45 hits / year for the last ten years (Figure 17). Within this period, we
observe a relative stability between the different combinations of pests and BCA (Figure 18).
The main part of the publications (56%) of the cases deals with the biocontrol of
phytophagous arthropods on which we will focus here. 42% of the papers deal with the
biocontrol of weed. In this case, BCA are for 57% of the cases phytophagous insects and for
41% fungi (data not shown).
More than 70 arthropod pests were listed which cover 7 orders and approximately 40
families. As shown in Figure 19, Hemiptera and Lepidoptera were the two main orders with a

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total of 66% of the pest species and 70% of the publications. If the citation rate / order is
highly correlated with the number of pests / order, this trend hides a great variability at the
infra-order level. Indeed, the citation rate highly differs with regard to the pest species with a
median of 2 papers / pest species and a range from 1 to 13 citations. The 13 most cited
pests are listed in Figure 20. Two main observations can be drawn from this short list.
 Firstly, this list is quite equally composed of either very specialist pests like
Phyllocnistis citrella (on Citrus species), Mononychellus tanajoa (on cassava) or Toxoptera
citricida (on Citrus species) or more generalist taxa like Homalodisca vitripennis, Lymantria
dispar or Pseudococcus viburni. All of them are phytophagous pests whose damage are
linked either to their herbivory, consumption of sap or virus transmission except the
particular case of the fire ant Solenopsis invicta which is responsible for direct nuisance on
farmers or indirect ecological modifications in the agrosystems.
 The second observation is that the percentage of ClBC related publications / pest
is negatively correlated with the corresponding total number of references (including also
studies on other pest control strategies and/or various biological topics). For instance, 22%
of the 32 references focusing on H. vitripennis explicitly deal with classical biological control
while this percentage falls down to only 1% to 3% for well documented species like L. dispar,
S. invicta or D. virgifera virgifera. This may be explained by the fact that ClBC is mainly
considered as a “pionneer” pest control strategy that are developed either soon after the
emergence of a new invasive pest or on “non biological model” for which the investigations
on other biological aspects are limited.
[Recommendation 2 (Major – Politics, Scientist): Although Classical Biological
Control can be perceived as a “pioneer” pest control strategies on non “biological models”,
substantial investments are required on several biological aspects (e.g. community ecology,
population genetics)]
4.1.3.4. Biocontrol agents used
The biocontrol agents related to ClBC (hereafter ClBCA) against arthropod species
were not detailed in only 12% of the papers. These are in most of the cases either
prospective works (55%) such as faunistic inventories of natural enemies on “new” pests like
Diabrotica virgifera virgifera or retrospective studies (35%) on advanced programmes that
take into account several BCA (see Appendix 18.1). Among the documented cases, 76% of
ClBC programmes were based on the use of insect parasitoids (see Section 3.1.3.5).
Pathogens and nematodes on one side and predatory arthropods on the other side are
equally represented with about 12% of the publications for each case.
 Pathogens and Nematodes as candidate for ClBCA
The particular cases of pathogens and nematodes have been recently reviewed by
Hajek and co-workers (62, 632
). Our own survey indicates that half of the papers actually
deal with entomopathogenic fungi. Six pest species were identified including two mites
(Aceria guerreronis and Mononychellus tanajoa) and two insects (Aphis gossypii and
Coptotermes formosanus). However, except for the evaluation of Neozygites species against
M. tanajoa (14, 39, 42, 43), other attempts seem to be rather limited. With regard to the
catalogue of Hajek et al.(62), two other cases of entomopathogen fungi were missed in our
own survey. These are the introductions of Entomophaga maigmaiga and Metarhizium
anisopliae, against respectively the Lymantria dispar and the Curculionidae Otiorynchus
nodosus for which the sources of Hajek and coworkers were mainly personal
communications. The rather limited use of entomopathogenic fungi in ClBC was also
confirmed by the review of Shah and Pell(156). The use of viruses as biocontrol agent for
ClBC against arthropod pests were only documented fort three cases that are the
Lepidoptera species Anticarsia gemmatalis (48, 127) and Lymantria dispar (16) and the
2
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Coleoptera Oryctes rhinoceros (81). Microspodia as candidate for ClBC were reported in
only two studies (25, 165). The sole case of the use of nematodes is the study of Hurley et
al. (79) who studied the extension of the use of parasitic nematode Deladenus siricidicola
against the woodwasp Sirex noctilio.
 Predatory arthropods as candidate for ClBCA
The literature about predatory arthropods is dominated by four case-studies. The first
one is the classical biocontrol of the cassava green mites M. tanajoa by Typhlodromalus
aripo and, to a lesser extent, T. manihoti. All these studies are the extension of a very large
classical biocontrol programme at a continental scale; two main issues were addressed
during the recent decade that are the introduction and field evaluation of T. aripo in
Mozambique and Malawi (125, 194) and the ecological interactions with other species (14,
124, 193) or plants(55). The second case-study is those of the predatory ladybird Harmonia
axyridis (19, 90, 91, 137). The main concern of these publications is nevertheless not the
Research and Development in ClBC but rather the risks of non-intended effects and
geographic spray of this insect that is now considered as a world-wide invasive species.
Another case of the use of ladybird is those of Cryptolaemus montrouzieri and Scymnus
coccivora which have been successfully used to control the hibiscus mealybug
Maconellicoccus hirsutus (51, 86, 103) which is the extension of a worldwide use of these
species. The fourth main case-study is the classical biocontrol programme of Prostephanus
truncatus, a serious pest of stored maize beetle using Teretrius (formerly Teretriosa)
nigrescens (73, 169, 170). The lasts reported uses of predatory arthropods as candidate for
ClBC were those of the Coleoptera Laricobius nigrinus against the adelgid Adelges tsugae
(197) and the phytoseid Neoseiulus baraki against the coconut mite A. guerreronis (119).
Contrary to other cases which were the continuity of older programmes, these two studies
are associated with new BCA inventories undertaken during the last ten years - see
respectively (196) and(99).
4.1.3.5. Insect parasitoids as BCA
 Related journals papers and categorization of the studies
125 publications were used for this analysis. Only 14% were associated to
proceedings of meetings or other supports than journals. 43 different journals were identified
but 50% of the publications were published only by five: Biological Control (21%), BioControl
(8%), Biocontrol Science and Technology (7%), Florida Entomologist (7%) and Bulletin of
Entomological Research (7%). Impact Factors are respectively 1.805, 1.957, 0.874, 0.886
and 1.415.
The types of the works were categorized according to the simplified sequential steps
in R&D of biological programmes: BCA Inventories BCA characterization (systematic,
molecular tools) Pest or BCA rearing  BCA biology (life history traits, thermal biology,
behavioural ecology)  Pre-release survey  BCA introduction  Post-release survey.
Studies related to “non-target effects” (i.e. the direct or indirect impacts of the ClBCA on non-
target species) as well as those related to the “biocontrol disruption” (i.e. the negative
impacts of organisms on the ClBCA) (details in Appendix 18.3) were also categorized. As
shown in Figure 21, most of the ClBC related publications logically deals either with BCA
biology, BCA introductions or post-release surveys which are central steps of the ClBC
programmes. A strong discrepancy nevertheless exists between the different types of work
in term of scientific publication; highest Impact Factors are relied to studies linked to Non-
intended effects, Biocontrol disruption or BCA Biology.
[Recommendation 3 (Minor – Politics, Scientists): The different steps of R&D in
Classical Biological Control are currently unequally promoted with regard to “scientific
criteria”, with a clear emphasis on community ecology including non target effects. Such

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trend may be detrimental to the short-term development of less gratifying tasks and
consequently on the whole dynamism of ClBC.]
 BCA Introductions
As shown in Table 19, 65 introductions were recorded during the period of 1991-
2006. This list is probably not exhaustive insofar as “cryptic introductions” may have been
missed. This list does not also cover all the R&D in classical biocontrol programmes since
some programmes may have been interrupted before releases. A faunistic inventory of the
natural enemies of the North American leafhopper Scaphoideus titanus has for instance
been led by our lab in 2000-2002 but the rearing of BCA candidates (mainly dryinids and
egg-parasitoids) were not successful.
 All these releases involve 55 different biocontrol agents (all hymenopteran except
the Pseudacteon species used against the fire ant Solenopsis invicta) and 35 pests. 57% of
these pests were Hemiptera, other being quite equally distributed between Lepidoptera,
Diptera, Hymenoptera and Coleoptera.
 Most of these introductions were realized against pest found on orchards and in
particular Citrus. Other targeted crops were mainly tropical productions, ornamental or
forest.
 Most of the BCA introductions (42%) were realized in Europe or neighbouring
countries (including Mediterranean Basin) and in North America (26%). The percentages of
introductions in other geographical areas were: Australia-New Zealand and neighbouring
islands (12%), South America (8%), sub-Saharan Africa (8%), Pacific Islands (3%), Asia
(1%).
 The total number of released parasitoids and number of sites were highly variable
ranging respectively from 456 to 660000 individuals and from 2 to 132 sites. The percentage
of establishment was 83% and, when established, high parasitism was found in 42% of the
cases. It is noteworthy that these values are relatively high compared to other estimates and
we are currently unable to say if this is linked to an improvement of practices or
methodological differences or biases.
[Recommendation 4 (Major – Politics): With regard to natural or other human-
mediated introductions of exotic species, species flow associated with the ClBC seems to be
rather limited. Although possible non-intended effects cannot be excluded (their studies
having to be increased), we fear that too drastic regulations could severely disturbed R&D
programmes]
[Recommendation 5 (Major – Scientists): Estimating the success of ClBC is difficult
because of methodological several biases (“cryptic introductions”, barriers linked to
languages and/or publishing). Shared international database should be necessary for more
accurate estimation as well as an increasing traceability.]
[Recommendation 6 (Minor –Scientist)]: In parallel with the geographical expansion
of their related pests, some biocontrol agents have been repeatedly released and
established worldwide. Population genetics studies in such pest-BCA interactions should be
particularly interesting to understand local adaptations, co-evolutionary processes and
ultimately, the durability of Classical Biological Control.]

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4.2.Currently registered biocontrol products in the EU
4.2.1. Collection of information
A small team formed by ACTA and IBMA conducted a survey on biological active substances
approved in the European Union and on Biological Control Products (BC products) authorised in a
several countries. The investigation focused on crops covered by ENDURE RA1case studies. The
frame of the present survey was defined in a meeting on 9th
January 2009 in Basle, and the work was
performed during the period from April to September 2009.
To compile a list of registered biocontrol products, the online EU Pesticides Database was consulted
on 21st
April 2009. Data were retrieved and the list was reorganised and the information about use
categories complemented with the help of the inclusion directives where necessary. Substances
deemed suitable for biocontrol were identified and it was decided to distinguish four major groups:
micro-organisms, semiochemicals (attractants), botanicals and "other plant protection substances of
natural origin".
This study was complemented by an analysis of specific uses of products commercialized in four
countries of the EU (France, Germany, Spain and the United Kingdom). A fifth country, Switzerland
was included in the study for comparison, because it has not been restricted by the implementation of
Directive 91/414/EEC until recently. For each country, official national online databases on authorised
plant protection products (Table 20) were screened for authorised biocontrol active substances:
Table 20: Consulted sources of information on authorized biocontrol plant protection products in five
European countries:
Country Official source / website Reference date
France
e-phy database of the Ministry of Agriculture & Fisheries
http://e-phy.agriculture.gouv.fr
31/8/2009
Germany
Online-Datenbank Pflanzenschutzmittel of the Federal Office of Consumer
Protection and Food Safety (BVL)
http://www.bvl.bund.de/cln_027/DE/04__Pflanzenschutzmittel/02__Zugel
assenePflanzenschutzmittel/02__OnlineDatenbank/onlineDB__node.html_
_nnn=true
12 /8/2009
Spain
Registro de productos Fitosanitarios of the Ministerio de Ambiente y
Medio Rural y Marino
http://www.mapa.es/es/agricultura/pags/fitos/registro/menu.asp
Switzerland
Plant protection index ("Pflanzenschutzmittelverzeichnis") of the Federal
Office for Agriculture (BWL, Fachbereich Pflanzenschutzmittel)
http://www.psa.blw.admin.ch/index_de_5_2_A.htm
31/7/2009
United
Kingdom
Pesticides Register of UK approved products under the responsibility of
the Chemicals Regulation Directorate Pesticides
https://secure.pesticides.gov.uk/pestreg/ProdSearch.asp
4/2009
The survey was limited to uses concerning seven crops or cropping groups which are subject to
ENDURE case studies: pomefruit (apples and pears), grapevine, cereals, rape, maize, potatoes and
tomatoes (greenhouse and field), the latter being extended to other vegetables where deemed of
interest. Country lists of representative products (generally up to two) were created and sorted
according to uses in crops, target pests and pathogens were identified by English and scientific names
wherever possible.

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4.2.2. Substances suitable for biological control and registered on Annex 1 of
the EU Pesticides Database
The complete list compiled from data retrieved in the EU Pesticides Database is presented in
Appendix 19. Excerpts concerning the four categories of substances compatible with biological control
are presented in Table 21.
4.2.2.1. Botanicals
Botanicals are plant-substances resulting from simple processing e.g. pressing or from extraction. By
extension the definition applies to a small numbers of compounds or even single ones extracted from
plants and purified e.g. laminarine.
Fourteen botanicals have been identified (Table 21) including two borderline cases for which single
molecules identical to naturally occurring substances have been synthesised.
- Four botanicals are authorised as repellents only: Extract from the tea tree, garlic extract, clove oil
(plant oils) and pepper.
- Six botanicals enter into the category of plant growth regulators.
- The phytohormones gibberellic acid and gibberelline are botanicals produced in fermenters acting
on plant growth. Spearmint oil and sea-alga extract are listed for their effect on plant growth as
well.
- The phytohormone ethylene is naturally present in plants and in soil and can be included here
although it is typically produced in the petrochemical industry by steam cracking.
- Carvone is a terpene produced by aromatic plants in particular by the mint it can also be classified
among the botanicals. To obtain a pure grade it is generally synthesised. In plant protection it is
used as a growth regulator.
- Laminarin is extracted from sea weed and is classified as elicitor. Rape seed oil enters into the
category of insecticides/acaroids. Citronella oil is the only BCA approved as a herbicide.
- Pyrethrins are extracted from Pyrethrum flowers, from cultivars of Chrysanthemum
cinerariaefolium. By their origin they are botanicals but their structures are analogue and their
properties are similar to those of synthetic pyrethroids. Due to their mode of action which is
analogous to conventional insecticides and their toxicity for aquatic and other non target
organisms, they are not typical biological substances although they are accepted in organic
farming.
Table 21: Active substances suitable for biological control listed on Annex I of 91/414/EEC (EU
Pesticide Database) - Status on 21st April 2009
Substance Category
1, 2
List
3 Inclusion
Date
Expiry Date Legislation
Botanicals
Extract from tea tree RE A 4 01/09/2009 31/08/2019 2008/127
Garlic extract RE A 4 01/09/2009 31/08/2019 2008/127
Gibberellic acid PG A 4 01/09/2009 31/08/2019 2008/127
Gibberellin PG A 4 01/09/2009 31/08/2019 2008/127
Laminarin EL C 01/04/2005 31/03/2015 05/3/EC
Pepper RE A 4 01/09/2009 31/08/2019 2008/127
Plant oils / Citronella oil HB A 4 01/09/2009 31/08/2019 2008/127
Plant oils / Clove oil RE A 4 01/09/2009 31/08/2019 2008/127
Plant oils / Rape seed oil IN, AC A 4 01/09/2009 31/08/2019 2008/127
Plant oils / Spearmint oil PG A 4 01/09/2009 31/08/2019 2008/127
Sea-algae extract (formerly sea-algae extract and PG A 4 01/09/2009 31/08/2019 2008/127

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fungi, protozoa, viruses and viroids. It does not include multicellular organisms, such as nematodes or
insects.
Twenty five microbial species are included in annex I, some of which are represented by several
strains.
Six bacterial (sub)species (Bacillus subtilis, Pseudomonas chlororaphis and four subspecies of
Bacillus thuringiensis) and two virus species (Cydia pomonella Granulose Virus and Spodoptera
exigua NPV) are included. All B.t. subspecies and viral agents are approved for insect control.
Pseudomonas is approved for fungicidal seed treatments and Bacillus subtilis can be used against
plant pathogenic fungi and bacteria.
Seventeen fungal agents belonging to twelve genera are listed, Trichoderma being represented by five
species. Beauveria bassiana, Lecanicillimum muscarium and Metarhizium anisopliae are approved for
use as insecticides, the other fungal agents for use against fungal diseases.
4.2.2.3. Semiochemicals (attractants)
Semiochemicals are chemical substances such as pheromones, kairomones and allomones that act to
modify the behaviour of pests or their natural enemies.
In the table based on the EU Pesticides Database, Straight Chain Lepidopteran Pheromones (SCLP)
are highlighted in green, non SCLP-pheromones in light cyan and other attractants (including
hydrolysed proteins) are highlighted in yellow. There is one repellent which is marked in light red.
SCLPs are included in annex I as a group but 25 compounds of this group are also listed individually.
In the inclusion directive 2008/127/EC, some molecules are mentioned three times, as an individual
substance, in a blend of the same type, e.g. acetates and in mixed blends, e.g. alcohols and acetates.
Often single SCLP compounds show attraction to one or more moth species and typically a
combination of two or more of these compounds in a precise ratio enhances the attraction and the
specificity. Thus SCLPs should be considered as a whole group and it must not be concluded that
each compounds stands for one species.
The SCLPs listed individually are typical examples found in the pheromone blends of moth pest
species currently of economic importance. A large variety of compounds and isomers, an estimated
number of about 300 identified molecules, used by Lepidopterans are not listed here. They differ in
carbon chain length, in the number of double bonds and/or their positions and in their chemical
functional group (alcohol, acetate or aldehyde)
SCLPs can be used for mass trapping, mating disruption or in attract and kill devices (A&K) or
formulations. When associated with an insecticide, i.e in A&K devices, attractants do not need to be
included in annex I.
Two non SCLP pheromones as well as four semiochemicals other than pheromones attractive to
different fly (Diptera) species are listed in the EU Pesticides Database: Ammonium acetate,
hydrolysed proteins, putrescine (1,4-diaminobutane) and Trimethylamine hydrochloride.
4.2.2.4. Other Plant Protection substances of natural origin
This group has been created for the purpose of the survey. It includes mineral substances as well as
substances produced by or derived from animals or from micro-organisms. Thus very diversified
substances and products like limestone powder, kaolin as well as diatomaceous earth (Kieselguhr),

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fatty acids and their derivates (e.g. soaps) can be found in this group. Not all substances of this group
do meet the expectation of low non-target toxicity and low environmental impact.
Some active substances included in annex I are produced by micro-organisms. Spinosad which is
produced by the bacterium Saccharopolyspora spinosa finds its place here; it is accepted for organic
farming. Milbemectin is a mixture of natural compounds (milbemycins) isolated from fermentation
broth of the fungus Streptomyces hygroscopicus subsp. aureolacrimosus. The substance is active
against insects of different families and a large range of mites. Abamectin contains avermectins which
are biosynthesised by Streptomyces avermitilis. The substance shows very high toxicity in Mammals
and in aquatic organisms. Milbemectin and abamectin are not authorised in organic crop protection.
Potassium hydrogen carbonate is a slightly basic substance used for its fungicidal properties. The US
FDA considers this substance as GRAS (Generally Recognised as Safe).
Six natural substances are specifically marked in the EU List, they are used as animal repellents:
three are minerals (Calcium carbonate, limestone, sodium aluminium silicate), two are of animal origin
(fish oil and sheep fat) while methyl nonyle ketone is either produced by synthesis or extracted from
plant oils (rue). The latter repellent acts by its strong odour. It is naturally present in some edible crops
and spices.
 Limit cases and exclusions
With regards to their (eco)toxicological profile and environmental impact neither sulphur and its
derivates (iron sulphate) nor cupric compounds i.e. Bordeaux mixture, copper hydroxide, copper
oxichloride and cuprous oxide are considered here as typical biological substances although they
might be accepted in organic agriculture.
Tall oils (crude or pitch) are a by-product in the Kraft process used in the paper industry. Thus they are
substances resulting from a chemical process and are classified as chemicals here.
Calcium carbide is produced from lime and coke in electric arc furnaces. It is fitted among chemicals
but is used as a repellent like some other minerals.
1-Methyl-cyclopropene is an inhibitor of the effects of the phytohormone ethylene and is mainly used
to conserve cut flowers. It is placed among the chemicals.
4.2.3. Uses of biocontrol products in five European countries
4.2.3.1. Registered plant protection substances
In each country all BCAs authorised for uses in seven crops or cropping groups were identified. Lists
of representative products (generally up to two) were created and sorted according to uses in crops:
pomefruit (apples and pears), vine, cereals, rape, maize, potatoes and tomatoes (greenhouse and
field), the latter was extended to other vegetables where deemed of interest.
In France twelve different microbial BCA species (or sub-species in the case of Bacillus thuringiensis)
are authorised among which two species, Beauveria tenella and Candida oleophila are not yet
included in 91/414 Annex I. Only four botanical active substances are authorised, including pyrethrum
and rotenon which were excluded from our survey. Fenugreek extracts benefited from a specific
French approach to plant extracts under former national rules, and EU approval for this active is still
pending. Laminarin is included in Annex I. Five Straight Chain Lepidopteran Pheromones (SCLP)
blends or associations (one just specifying minor components used for the single target codling moth)
are registered for mating disruption in orchards or vineyard.

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In Germany nine microbial BCAs are authorised in Plant Protection Products (all included). Only four
botanical substances are listed for plant protection, two of which are included in Annex I (pyrethrins
and rape seed oil), two are not (azadirachtin and lecithin). Three different SCLP associations are
authorised for mating disruption against Codling Moth or Vine Moths.
For Germany only fully registered BC products according to the rules of the PPP directive were
included in the survey. As a consequence, plant strengtheners authorised according to the Federal
Plant Protection Act §§ 31ff were excluded. Plant Strengtheners can avoid the EU procedures and
requiremments for plant protection products but they must not claim specific protective properties
either.
In Spain ten microbial BCAs are authorised, all of which are included in EU Annex 1. Only three
botanical substances could be identified: Pyrethrins and rotenon which are excluded from the survey
and Azadirachtin (Neem extract) which is not included in EU Annex I. The plant growth regulators
gibberellinic acid/gibberellin were not explored. Only four SCLP associations are authorised for mating
disruption in vine and orchards including two for oriental fruit moth and peach twig borer typical for
peach orchards.
In Switzerland twelve different microbial BCA species (or sub-species in the case of Bacillus
thuringiensis) are authorised, among which is one species not included in 91/414 Annex I: Beauveria
brognartii.
Eleven botanicals are approved, among which the insecticides Pyrethrum (included in EU Annex I)
and rotenon (rejected from Annex I) have been excluded from the survey because of their toxicological
profile. The plant growth regulators gibberellic acid and gibberellin were also excluded from the
survey. Five substances not included in EU Annex I are authorised: Azadirachtin (Neem extract),
fennel oil, lecithin, mustard powder and Quassia extract.
An impressive number of semiochemicals, eleven different SCLP associations are authorised for
mating disruption allowing the control a large variety of moths in orchards (including one association of
8 compounds against five different species) and vineyards. This can be related to the facilitated
approval of pheromone products in Switzerland.
In the UK eight microbial BCAs are approved but only a single botanical (Laminarin, EU approved)
and a single pheromone blend (for codling moth). No biological plant protection products are available
for use in grapevine, rape, maize or potatoes.
With regard to the global availability of biological control products in the different crops, pomefruit,
vegetables and vine are generally in a better position than arable crops in the countries included in the
survey. In the UK e.g. only laminarin is available on wheat and cereals, and no biological plant
protection products are registered for rape, maize or potatoes.
Conclusion: None of the EU Member States covered in the present survey shows such a variety of
BCAs as Switzerland where we find the largest numbers of microbials, botanicals and pheromone
blends authorised in the crops subject of the inquiry. Only France reaches the number of twelve
microbial BCAs in registered products. The privileged situation in the Helvetic Confederation can be
explained by the flexible regulatory approach of the competent authorities in the past, until the
progressive implementation of EU directive 91/414/EEC and the related framework, as well as the
sustained support by experts in confederal agronomic institutes.

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4.2.3.2. Invertebrate BCAs
Invertebrate BCAs were listed separately for each country.
In France invertebrate BCAs cannot be registered, they do not even need to be formally declared. The
list provided in the survey is based on the voluntary declarations to ACTA by the producers wishing to
have their beneficials published in the non-official Index Phytosanitaire.
They must be registered in Germany. An official list which is regularly updated is published by the
Julius Kühn Institute.
In Spain companies which are responsible of commercialisation of IBCAs must give an information to
the Ministry of Agriculture to allow the inscription into a register before commercialisation (Orden
APA/1470/2007). This information given is about name of commercial product, identification of the
organism, the manufacturer, the responsible of commercialisation. Another law (43/2002; 20th
of
November 2002) covers the introduction of exotic organisms (article 44).
In Switzerland invertebrate BCAs must be formally approved by the BLW (Bundesamt für
Landwirtschaft) and they are listed together with the plant protection products.
In the UK no authorisation is required to release indigenous beneficials but the import (and release) of
non indigenous species must be approved by the Advisory Committee for the release of exotics
(ACRE acting under DEFRA).
4.3.Regulatory aspects
4.3.1. Objectives
The objective of the work was to identify typical hurdles for the placing of biological plant protection
products on the market experienced by biocontrol industry or evaluators in the recent past under the
European directive 91/414/EEC. In parallel, we examined the new regulation (No 1107/2009/EC of
the European Parliament and of the Council of 21 October 2009) concerning the placing of plant
protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC and
the new directive (N° 2009/128/EC of the European Parliament and of the Council of 21 October 2009)
establishing a framework for Community action to achieve the sustainable use of pesticides. These
two texts1
were examined for provisions creating new opportunities for the approval biocontrol agents,
their placing on the market and use. In fine, it was the intent to establish a dialogue with EU regulators
and evaluators in European institutions, i.e. in the European Commission and in the European Food
Safety Agency (EFSA) and to seek solutions in common for the problems encountered.
4.3.2. Working method
An ad hoc group of representatives from biocontrol industry and INRA called "Regulatory Review
Team" was set up. Two full-day working sessions were organised on 12th
March in Paris and on 18th
May in Basle in which regulatory experts identified difficulties and questions but also described
positive experience and perspectives.
The work of the Regulatory Review Team active under RA4.3 was summarised and reported in a
meeting of a delegation of ENDURE partners (IBMA, INRA and ACTA) with representatives of the
1
The text is available form the Official Journal of the European Union L 309, Volume 52 of 24th November and can be downloaded
from the EUR-Lex website: http://eur-lex.europa.eu/JOHtml.do?uri=OJ%3AL%3A2009%3A309%3ASOM%3AEN%3AHTML .

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European Commission (DG SANCO, DG Agriculture, DG Research) and the EFSA on 24th
September in Brussels.
4.3.3. Results
PowerPoint files of the presentations in the working sessions on general regulatory issues (U.
Heilig/IBMA), micro-organisms (C. Alabouvette/INRA & chair of the CES on Micro-organisms giving
opinion to Afssa-DiVE), Straight Chain Lepidopteran Pheromones (R. Sheppard/ IBMA) and
Botanicals (N. Walther/IBMA) are available in the restricted access section of the ENDURE website.
The PowerPoint presentation under the title "Gaps - Problems - Opportunities for BCAs in E.U.
Regulation - From Past to Future" prepared for the ENDURE – Commission meeting can also be
accessed there. In this final document, two key issues related to directive 2001/36/EC annex II B fixing
requirements for microbial active substances were highlighted. Tests suggested by evaluation
experts and intended to establish the genetic stability of a strain do not reflect practical conditions,
while in the case of potential microbial contaminants no European reference list is available. The
incidence of many pathogens can be excluded by production methods or the geographic location of
production sites. Tolerance limits for contamination levels could take into consideration thresholds
used in food industry, application levels for the microbial product and naturally occurring background
levels. The two issues presented here but also other examples put forward to the Regulatory Review
Team lead to the statement that "not all the studies or tests that can be performed for microbials will
necessarily yield relevant data".
The most important experience with semiochemicals was made during the on-going re-assessment of
Straight Chain Lepidopteran Pheromones (SCLPs), which were supported by an IBMA Task Force.
Regulators and evaluators were flexible in accepting a single common dossier for all compounds
notified but although an OECD guidance document recommends data waiving for numerous SCLP
requirements, the Rapporteur Member State insisted that all existing data and study reports on all
compounds be submitted on the grounds that the requirements of the directive are superior to the
guidance document recommendations. So far, the re-assessment procedure resulted in the inclusion
with postponed peer review of SCLPs as a group, but 25 substances are also listed individually. New
substances can be included in a simplified procedure provided that the applicant has access to the
existing dossier. Remaining questions include what industry input will be required during the peer
review by EFSA, the E.U. status of a revised OECD guidance document for semiochemicals other
than SCLPs, the decision if MRLs are required for sprayable SCLP formulations, and equivalence
criteria for SCLP substances. It was also noted that under the Biocidal Product Directive, rules and
fees applied to SCLPs created an economic hurdle which resulted in the submission of a dossier for
only one compound.
Extracts from plants - as long as not purified - consist of mixtures of molecules while data
requirements of directive 91/414/EEC are basically designed for defined single substances. Thus
those requirements often do not fit for mixtures of several substances. It must be decided if the most
“active” substance, the one with the highest content in the extract or the whole extract shall be used in
studies required for different sections of a dossier i.e. for data on physical-chemical properties,
metabolism, toxicology, residues, environmental fate and behaviour, and which data shall be used in
risk assessment. While the whole extract can be recommended for use in toxicity studies, it is not
convenient for residue, metabolism or environmental studies because in practice it is generally not
possible to determine the fate of all compounds contained in an extract. Questions asked by
evaluators from several Member States after the issuing of a draft assessment report for Neem extract
and its lead substance Azadirachtin A illustrate the difficulties experienced by an applicant in the
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The new regulation concerning the placing of plant protection products on the market provides for a
specific status for "low risk active substances" (article 22). Many biocontrol substances can be
expected to qualify for this new category but one exclusion criterion, the half-life in soil, may cause
problems for microbial active substances unless it is clearly limited to chemicals. A full set of data is
required to gain the status of low risk active substance but products containing them exclusively and
without co-formulants of concern will benefit from reduced dossier requirements and time lines for
approval. Micro-organisms, plant extracts or other natural substances may also meet the criteria for
"Basic substances" provided for in article 23 but the discussion in the ENDURE-Commission meeting
made it clear that this category is without interest for manufacturers who intend to market their
substances for plant protection. It was noted that the new regulation does not provide for generic
waivers i.e. for justifications of non submission of data or exemptions from requirements for groups of
substances or products.
In the framework directive a number of provisions in favour of biological pest control measures or
non-chemical methods have been identified. The new regulation also mentions in recital 35 that
priority should be given to "non-chemical and natural alternatives wherever possible" but since the
definition of non-chemical methods refers to "physical, mechanical or biological pest control" and does
not specifically mention microbials, semiochemicals, botanicals or other natural substances with non-
toxic mode of action it must be clarified how those groups are covered by the definition.
4.3.4. Conclusion
In the meeting between the ENDURE delegation and representatives of the European
Commission, the need for discussions between regulators, evaluators and industry about
requirements especially those relevant for microbial and botanical substances was recognised. Article
77 of the new plant protection product regulation authorises the Commission to "adopt or amend
technical and other guidance documents e.g. explanatory notes or guidance documents on the
content of the application concerning micro-organisms, pheromones and biological products." Thus at
least part of the problems experienced by applicants can be addressed in guidance documents.
Industry representatives and companies directly concerned by evaluations or reviews of biocontrol
agents should enter into discussions with evaluators (EFSA or Competent Authorities in Member
States) without forgetting the leading role of the Commission. Industry should fix priorities, prepare
rationales and make substantiated proposals dealing with data requirements considered inappropriate,
unnecessary or unrealistic.
4.4.Identified difficulties and conditions for success at field level
4.4.1. Technical aspects: factors of efficacy
4.4.1.1. Quality of the BCAs formulations
Numerous investigations on the development of biopesticides have been initiated as legislation and
government policy have demanded less reliance on chemical pesticides and greater adoption of IPM.
In Europe, some countries have set goals of reducing pesticide use by 50%. Successes have been
achieved through better timing of applications, so that lower dosages are effective and substituting
less hazardous and more active materials, to reduce the number of applications.
Biopesticides are distinguished from conventional chemical pesticides as many are very selective and
are non-toxic towards non-target organisms. While biopesticides are likely to be less harmful to the
environment than the conventional ones, care needs to be taken that wastage is minimised, by
selecting the most appropriate droplet spectrum. A disadvantage of biological agents relative to
chemicals, is that many are not sufficiently persistent and are relatively slow acting; therefore,
research has been directed at extending the period of activity. However, some such agents may

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persist in the field or the forest for many months, and a risk–benefit analysis should be performed to
establish their environmental acceptability.
Transition from the optimised conditions of a laboratory experiment to the harsh conditions
experienced in the field has so far proved more difficult for application of biopesticides in contrast to
chemicals. This has undoubtedly been due to lack of investment in the development of effective
formulations and delivery systems, in order to commercialise more potential biopesticides. The
relatively small effort invested in target-specific sprayers, compared with the investment in laboratory
studies, has led to unbalanced development, and exemplifies the need for closer integration between
formulation and engineering research. The challenge is to get effective formulations so that biological
control agents can be easily applied by farmers.
4.4.1.2. A good example, the case of Trichoderma: direct and indirect mode of
action against plant pathogens
Trichoderma species have long been recognized as biological control agents (BCAs) for the control of
plant disease and for their ability to increase plant growth and development. They are widely used in
agriculture, and some of the most useful strains demonstrate a property known as ‘rhizosphere
competence’, the ability to colonize and grow in association with plant roots (Harman, 20001
). Much of
the known biology and many of the uses of these fungi have been documented recently (Kubicek et
al., 1998, Harman et al., 2004c, Perello et al., 2009). The taxonomy of this fungal genus is continually
being revised, and many new species are being described (Komon-Zelazowska et al., 2007);
(Samuels, 2006, Overton et al., 2006, Kubicek et al., 2008, Samuels and Ismaiel, 2009). The
mechanisms that Trichoderma uses to antagonize phytopathogenic fungi include competition,
colonization, antibiosis and direct mycoparasitism (Howell, 2003, Harman, 2006). This antagonistic
potential serves as the basis for effective biological control applications of different Trichoderma
strains as an alternative method to chemicals for the control of a wide spectrum of plant pathogens
(Harman et al., 1991).
The colonization of the root system by rhizosphere competent strains of Trichoderma results in
increased development of root and/or aerial systems and crop yields (Chacon et al., 2007); (Kubicek
et al., 1998); (Yedidia et al., 2003). Trichoderma has also been described as being involved in other
biological activities such as the induction of plant systemic resistance and antagonistic effects on plant
pathogenic nematodes (Sharon et al., 2001, Jegathambigai et al., 2008).Some strains of Trichoderma
have also been noted to be aggressive biodegraders in their saprophytic phases, in addition to acting
as competitors to fungal pathogens, particularly when nutrients are a limiting factor in the environment
(Worasatit et al., 1994). These facts strongly suggest that in the plant root environment Trichoderma
actively interacts with the components in the soil community, the plant, bacteria, fungi, other
organisms, such as nematodes or insects, that share the same ecological niche.
Trichoderma spp. are important participants in the nutrient cycle. They aid in the decomposition of
organic matter and make available to the plant many elements normally inaccessible. Yedidia et al.
(Yedidia et al., 2001) noted that the presence of the fungus increased the uptake and concentration of
a variety of nutrients (copper, phosphorus, iron, manganese and sodium) in the roots of plants grown
in hydroponic culture, even under axenic conditions. These increased concentrations indicated an
improvement in plant active-uptake mechanisms. Corn that developed from seeds treated with T.
harzianum strain T-22 produced higher yields, even when a fertilizer containing 40% less nitrogen was
applied, than the plants developed from seed that was not treated with T-22 (Harman, 2000). This
ability to enhance production with less nitrate fertilizer, provides the opportunity to potentially reduce
nitrate pollution of ground and surface water, a serious adverse consequence of large-scale maize
culture. In addition to effects on the increase of nutrient uptake and the efficiency of nitrogen use, the
beneficial fungi can also solubilize various nutrients in the soil, that would be otherwise unavailable for
uptake by the plant (Altomare et al., 1999b).
1
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The cross-talk that occurs between the fungal BCA and the plant is important both for identification of
each component to one another and for obtaining beneficial effects. Somehow, the plant is able to
sense, possibly by detection of the released fungal compounds, that Trichoderma is not a hostile
presence, therefore the plant defence system is not activated as it is when there is pest attack and the
BCA is recognized as a plant symbiont rather than a plant pathogen (Woo and Lorito, 2006).
Molecules produced by Trichoderma and/or its metabolic activity also have potential for promoting
plant growth (Chacón et al., 2007); (Vinale et al., 2008a, Vinale et al., 2008b);(Yedidia et al., 1999).
Applications of T. harzianum to seed or the plant resulted in improved germination, increased plant
size, augmented leaf area and weight, greater yields (Altomare et al., 1999a, Harman et al., 2004a,
Harman et al., 2004b, Inbar and Chet, 1995, Vinale et al., 2008b).
Numerous studies indicated that metabolic changes occur in the root during colonization by
Trichoderma spp., such as the activation of pathogenesis-related proteins (PR-proteins), which induce
in the plant an increased resistance to subsequent attack by numerous microbial pathogens (Table
22).
Table 22: Evidence for, and effectiveness of, induced resistance in plants by Trichoderma species
(Harman et al., 2004a).
The induction of systemic resistance (ISR) observed in planta determines an improved control of
different classes of pathogens (mainly fungi and bacteria), which are spatially and temporally distant
from the Trichoderma inoculation site. This phenomenon has been observed in many plant species,

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both dicotyledons (tomato, pepper, tobacco, cotton, bean, cucumber) and monocotyledions (corn,
rice). For example, Trichoderma induces resistance towards Botrytis cinerea in tomato, tobacco,
lettuce, pepper and bean plants, with a symptom reduction ranging from 25 to 100%. Moreover,
Trichoderma determined an overall increased production of defence-related plant enzymes, including
various peroxidases, chitinases, β-1,3-glucanases, and the lipoxygenase-pathway hydroperoxide
lyase (Harman et al., 2004a, Howell et al., 2000, Yedidia et al., 1999) of T. harzianum strain T-39, the
active ingredient of the commercial product TricodexTM.
Thus far, Trichoderma is able not only to produce toxic compounds with a direct antimicrobial activity
against pathogens, but also to generate fungal substances that are able to stimulate the plant to
produce its own defence metabolites. In fact, the ability of T. virens to induce phytoalexin
accumulation and localized resistance in cotton has already been discussed (Hanson and Howell,
2004). In cucumber, root colonization by strain T-203 of T. asperellum caused an increase in phenolic
glucoside levels in the leaves; the aglycones, which are phenolic glucosides with the carbohydrate
moieties removed, are strongly inhibitory to a range of bacteria and fungi (Yedidia et al., 2003).
A fundamental part of the Trichoderma antifungal capability consists in the production and secretion of
a great variety of extracellular cell wall degrading enzymes (CWDEs), including endochitinases, β-N-
acetylhexosaminidase (N-acetyl-β-D-glucosaminidase), chitin-1,4-β-chitobiosidases, proteases, endo-
and exo-β-1,3-glucanases, endo β-1,6-glucanases, lipases, xylanases, mananases, pectinases, pectin
lyases, amylases, phospholipases, RNAses, DNAses, etc. (Benítez et al., 1998; Lorito, 1998). The
chitinolytic and glucanolytic enzymes are especially valuable for their CWDE activity on fungal plant
pathogens, hydrolyzing polymers not present in plant tissues (Woo et al., 1999). Each of these classes
of enzymes contains diverse sets of proteins with distinct enzymatic activities. Some have been
purified, characterized and their encoding genes cloned (Ait-Lahsen et al., 2001, de la Cruz et al.,
1992, de la Cruz et al., 1995a, de la Cruz et al., 1995b, Garcia et al., 1994, Limon et al., 1995, Lora et
al., 1995, Lorito et al., 1994a, Lorito et al., 1993, Montero et al., 2007, Peterbauer et al., 1996, Suarez
et al., 2004, Viterbo et al., 2001, Viterbo et al., 2002). Once purified, many Trichoderma enzymes have
shown to have strong antifungal activity against a wide variety of phytopathogens, and they are
capable of hydrolyzing not only the tender young hyphal tips of the target fungal host, but they are also
able to degrade the hard, resistant conservation structures such as sclerozi.
Trichoderma spp. have been widely studied, and are presently marketed as biopesticides, biofertilizers
and soil amendments, due to their ability to protect plants, enhance vegetative growth and contain
pathogen populations under numerous agricultural conditions (Harman, 2000, Harman, 2004, Vinale
et al., 2008a). The commercial success of products containing these fungal antagonists can be
attributed to the large volume of viable propagules that can be produced rapidly and readily on
numerous substrates at a low cost in diverse fermentation systems. The living microorganisms,
conserved as spores, can be incorporated into various formulations, liquid, granules or powder etc.,
and stored for months without losing their efficacy (Jin et al., 1996). To date more than 50 different
Trichoderma-based preparations are commercialized and used to protect or increase the productivity
of numerous horticultural and ornamental crops (Table 23; Lorito et al. 2006).

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4.4.1.3. The case Trichoderma: mode of application, persistence on the
target and new formulations.
Effectiveness under controlled conditions (even under field conditions) does not necessarily
guarantee that the organism will perform successfully; proper formulation is a prime condition for
meeting market requirements. For instance an efficient biocontrol agent of soilborne and airborne
pathogens must first and foremost protect the young seedling against detrimental attack by
infective inoculum. Therefore some factors may be considered:
(a) soil ecosystem factors such as moisture, pH, structure, and temperature; (b) root colonization
capacity;
(c) reasonable shelf life;
(d) efficiency of application of the biocontrol agent in terms of its specific habitat and target
(Spiegel and Chet, 1998)
Many preparations have been developed to ensure a good shelf life of the product based on
Trichoderma. Some of that formulation are stable in terms of pH, that remains constant and low
(5.5) during the entire growth period, thus preventing bacterial contamination. Moreover the shelf
life of the fungus at 25 °C is 1 year and from 1 to 2 years, the number of colonies-forming-units
(CFUs) decreases by one order of magnitude. Many of that formulation have been proven
successful in several experiments in the greenhouse and field. The rapid mass production of
promising antagonists in the form of spores, mycelia or mixtures of both, has been achieved by
liquid-fermentation technology: mass production of biomasses of T. hamatum, T. harzianum, and
T. viride was reached by utilizing commercially available, inexpensive ingredients such as
molasses, brewer's yeast, cotton seed flour, or corn-steeped liquor. 1984). Other techniques
have been employed to improve the delivery of the biocontrol agents. A lignite-stillage (a by-
product of sorghum fermentation) carrier system was tested for applying a T. harzianum
preparation to the soil. Encapsulation of the biocontrol agent in an alginate-clay matrix, using
Pyrax as the clay material, improved yield and propagule viability over time.
Pelletized formulations of wheat bran or kaolin clay in an alginate gel containing conidia,
chlamydospores or fermentex biomass of several Trichoderma isolates revealed increased
viability of stored pellets, and the number of CFUs formed after adding these pellets to the soil
was comparable to that formed from freshly prepared pellets. These growth media and delivery
systems for formulations
of biocontrol fungi show promise because they are able to introduce high levels (106-
1010
CFU/g)
of fungi into soils not steamed, fumigated, or treated with other biocides.
To enhance biocontrol efficacy, appropriate introduction of the antagonist into the
microenvironment appears to be crucial: formulations have been applied to seedlings prior to
planting or to seeds in furrows. Economic considerations have forced biotechnologists to improve
the application techniques: seed-coating, a technique involving minimal amounts of inoculum
was developed.
Increased biocontrol activity may be achieved by combining two types (or more, if possible) of
biocontrol agents, for example combining Trichoderma with a bacterium, or another beneficial
fungus. The combined activity of the antifungal compounds produced by both microorganisms
could expand the spectrum of pathogens controlled. In fact, in field trials combining T. koningii
with certain fluorescent pseudomonads, greater suppression of take-all disease and increased
wheat yield were achieved relative to plants treated with T. koningii alone (Duffy et al., 1996).
Delivery systems must ensure that biocontrol agents will grow well and achieve their
purpose. It is generally recognized that delivery and application processes must be
developed on a crop by crop and application by application basis. No general solutions exist,
and so biocontrol systems must be developed for each crop. It is very important to use the
organism properly and to have appropriate expectations. Any biocontrol organism will be
unable to protect seeds as well as chemical fungicides. However, it colonizes roots,
increases root mass and health, and consequently frequently provides yield increases, which
chemical fungicides applied at reasonable rates cannot do. An effective method of use is to
use the biocontrol fungus in conjunction with chemical fungicides. The chemicals provide
good short-term seed protection, and the biocontrol fungus provides long-term root

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protection. As a consequence, yields frequently are increased over use of the chemical
alone.
Some experiences evidence that Trichoderma spp. is also highly effective when applied to
blossoms or fruits for control of B. cinerea. Even low levels of the organism applied to
strawberry blossoms by bee delivery or by sprays of liquid formulations are effective. For
maximum control of the Botrytis bunch rot of grape, this initial application needs to be
augmented by sprays as fruits mature, and addition of iprodione as a tank mix to this late
application appears to have synergistic activity over either the biocontrol agent or the
chemical fungicide alone.
 Novel applications of Trichoderma spp.
Trichoderma produces a variety of lytic enzymes that have a high diversity of structural and
kinetic properties, thus increasing the probability of this fungus to counteract the inhibitory
mechanisms used by neighbouring microorganisms. Further, Trichoderma hydrolytic
enzymes have been demonstrated to be synergistic, showing an augmented antifungal
activity when combined with themselves, other microbial enzymes, PR proteins of plants and
some xenobiotic compounds (Fogliano et al., 2002, Lorito et al., 1994a, Lorito et al., 1996,
Lorito et al., 1994b, Lorito et al., 1994c, Lorito et al., 1998, Schirmbock et al., 1994, Woo et
al., 2002). In fact, the inhibitory effect of chemical fungicides for the control of the foliar
pathogen B. cinerea was substantially improved by the addition of minute quantities (10-20
ppm) of Trichoderma CWDEs to the treatment mixture (Lorito et al., 1994b).
Extensive testing of T. harzianum strain T22 conducted for the registration of this biocontrol
agent in the USA by the Environmental Protection Agency (EPA) has found that the CWDEs
do not have a toxic effect on humans and animals (ED50 and LD50), and that they do not
leave residues, but degrade innocuously in the environment. Therefore, these Trichoderma
hydrolytic enzymes present a novel product for plant disease control based on natural
mycoparasitic compounds used by the antagonistic fungi. Single or mixed combinations of
CWDEs with elevated antifungal effects, obtained from fermentation in inducing conditions,
over-expression of the encoding genes in strains of Trichoderma, or heterologous
expression of the encoding genes in other microbes are possible alternatives for pathogen
control. These natural substances originating from the BCA are an improvement over the
use of the living microorganism in the production of commercial formulations because they
are easily characterized, resist desiccation, are stable at temperatures up to 60° C, and are
active over a wide range of pH and temperatures in the agricultural environment.
 Some experiments conducted to evaluate concentration and stability
assessment of a new liquid Trichoderma bio-formulate.
In order to develop a marketable formulate, part of a culture broth obtained by fermentation
was concentrated by using spray drying and lyophilisation techniques. Glycerol was added
to the samples (20% v/v) to better preserve the spore vitality. Results showed in showed no
significant differences in terms of chitinolytic activity before after treatments. Moreover, spore
vitality was not significantly affected by the lyophilisation when glycerol was added; without
glycerol, the spore concentration reduced from 7.0 x 106
to 1.8 x 106
spores/ml after
treatment. Conversely, the sample treated by spray drying lost completely its activity and no
enzymatic activity was registered at all. To assess the stability of the novel formulate, the
decreases of spore vitality and enzymatic activities were monitored, as well as the effect of
different stabilizing compounds (ampicillin, mineral oil, glycerol, PMSF). The results showed
no considerable reduction of both spore vitality and chitinolytic activities at 45 and 110 d
after fermentation. Moreover, the different stabilizing treatments did not differ with each other
significantly (Ruocco personal communication).
The important factors to consider in a commercial bio-formulation are product stability, the
capacity to produce consistent results by preserving the characteristics producing the
biological effects; the storability of the material, the ability to be conserved in unspecialized

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conditions similar to those of chemical pesticides; and a reasonable shelf-life or time that the
product can be stored and used without compromising the efficacy (Agosin and Aguilera,
1998; Agosin et al., 1997; Powell and Jutsum, 1993). When a formulation contains the living
microorganism component, the treatment must consist of stabilizing the viability of the BCA.
For liquid formulations this can be achieved by maintaining the product in refrigeration (<10°
C) or by freezing in the presence of cryoprotectant substances. However, conservation of a
commercial product in these conditions is not economic for maintaining low temperatures or
efficient because the liquid is both bulky and heavy, plus it is difficult to sustain these
conditions in storage and transportation. In comparison, it is preferable to obtain formulations
that contain a dehydrated product, stored as a powder, granule, talc, etc. Some works
(Ruocco et al. unp) demonstrated that lyophilisation did not reduce chitinolytic activity and
spore vitality when the fermented cultures were treated with compounds that protect the
osmotic integrity of the living material such as glycerol. Generally, lyophilisation is the
method that best maintains viability, but its cost is very high. At the industrial level and in
order to obtain a low-cost product, the methods preferred is spray- or fluidized bed- drying.
Many products are obtained by spray-drying, but this method produces a high loss of viability
in some microorganisms (observed also in this formulation), due to the thermal treatment.
Moreover, different compounds (ampicillin, mineral oil, glycerol, PMSF) were added to
determine if they aided in to maintaining the stability of the formulation. The enzyme activity
in samples assayed over time were not affected neither positively nor negatively by the
addition of the compounds in comparison to the untreated control. Obviously, it is very
important to maintain good sanitary conditions throughout the fermentation process and
during packaging in order to avoid possible contamination that will compromise the product
during storage.
In spite of the relatively abundant number of patents filed for microbial pesticides, the
number of commercial applications has not been as dramatic as expected (Montesinos,
2003). In Europe, the limiting factor for registration, apart from the cost, is undoubtedly the
slow process of decision-taking. As an example, the first application for patenting a
biopesticide, Paecilomyces fumosoroseus, was submitted to the European Union in 1994
and approved only in 2001. In most cases, excessive specificity is a problem difficult to solve
because it is intrinsic to the biological control system. In fact, success depends on three
living systems: the pathogen or pest, the BCA and the host plant. Biosafety and
environmental concerns are also major limiting factors for microbial pesticide prospects.
Furthermore, the registration procedure to approve a biopesticide formulation on the market
has not been altered to consider the biological aspects of the product, criteria which are
different than those considered for the testing of chemical based products.
4.4.1.4. Persistence, physiological stresses, timing and coverage of
others biological agents
Others references have been screened for biocontrol agents considering the analysis of:
 persistence on the target,
 resistance to physiological stresses,
 timing and coverage.
 Cladosporium cladosporioides
The antagonist has been effective in reducing sporulation of Venturia inaequalis under
orchard conditions. Furthermore, the results of the pre-screening
indicate that it is cold and drought tolerant and results of experiments on spore production in
solid state fermentation show that mass production is economically feasible. These results
have been obtained in a stepwise selection approach (Kohl, 2009).
 Ulocladium atrum and Gliocladium roseum
Köhl et al., 1998 described the effect of treatments with conidial suspensions of Ulocladium
atrum and Gliocladium roseum on leaf rot of cyclamen caused by Botrytis cinerea was

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investigated under commercial greenhouse conditions. Spraying U. atrum (1 × 106 conidia
per ml) or G. roseum (2 × 106 conidia per ml and 1 × 107 conidia per ml) at intervals of 2 to
3 weeks during the production period and spraying U. atrum (1 × 106 conidia per ml) at
intervals of 4 to 6 weeks resulted in a significant reduction of natural infections of petioles by
B. cinerea. U. atrum or G. roseum (1 × 107 conidia per ml) was as effective as the standard
fungicide program. B. cinerea colonized senesced leaves within the plant canopy and
infected adjacent petioles and leaves later. The antagonists colonized senesced leaves and
reduced B. cinerea development on these leaves. Thus, the inoculum potential on petioles
adjacent to necrotic leaf tissues was reduced. The fate of U. atrum conidia on surfaces of
green cyclamen leaves during a 70-day period after application was studied. The number of
conidia per square centimetre of leaf surface remained relatively constant during the entire
experiment. Sixty percent of the conidia sampled during the experiments retained the ability
to germinate. When green leaves were removed from the plants to induce senescence and
subsequently were incubated in a moist chamber, U. atrum colonized the dead leaves.
Senesced leaves also were colonized by other naturally occurring fungi including B. cinerea.
On leaves treated with U. atrum from all sampling dates, sporulation of B. cinerea was
significantly less as compared with the untreated control. Our results indicate that early
applications of U. atrum before canopy closure may be sufficient to achieve commercially
satisfactory control of Botrytis leaf rot in cyclamen.
Kessel et al., 2005 developed a spatially explicit model describing saprophytic colonization
of dead cyclamen leaf tissue by the plant-pathogenic fungus Botrytis cinerea and the
saprophytic fungal antagonist Ulocladium atrum. Both fungi explore the leaf and utilize the
resources it provides. Leaf tissue is represented by a two-dimensional grid of square grid
cells. Fungal competition within grid cells is modelled using Lotka-Volterra equations. Spatial
expansion into neighbouring grid cells is assumed proportional to the mycelial density
gradient between donor and receptor cell. Established fungal biomass is immobile. Radial
growth rates of B. cinerea and U. atrum in dead cyclamen leaf tissue were measured to
determine parameters describing the spatial dynamics of the fungi. At temperatures from 5
to 25°C, B. cinerea colonies expanded twice as rapidly as U. atrum colonies. In practical
biological control, the slower colonization of space by U. atrum thus needs to be
compensated by a sufficiently dense and even distribution of conidia on the leaf. Simulation
results confirm the importance of spatial expansion to the outcome of the competitive
interaction between B. cinerea and U. atrum at leaf scale. A sensitivity analysis further
emphasized the importance of a uniform high density cover of vital U. atrum conidia on
target leaves.
4.4.1.5. References cited in Chapter 4.4.1
Agosin E, Volpe D, Munoz G, San Martin R, Crawford A (1997). Effect of culture conditions on spore
shelf life of the biocontrol agent Trichoderma harzianum. World J. Microbiol. Biotechnol., 13:
225-232.
Agosin E, Volpe D, Munoz G, San Martin R, Crawford A (1997). Effect of culture conditions on spore
shelf life of the biocontrol agent Trichoderma harzianum. World J. Microbiol. Biotechnol., 13:
225-232.
Agosin E, Aguilera JM (1998). Industrial production of active propagules of Trichoderma for
agricultural use. In: Trichoderma and Gliocladium. Volume 2, Enzymes, Biological control and
commercial applications, G.E. Harman and C.P. Kubicek eds., Taylor & Francis Ltd., London,
UK, pp. 205-227.
Ait-Lahsen, H., Soler, A., Rey, M., de La Cruz, J., Monte, E. & Llobell, A. (2001) An antifungal exo-
alpha-1,3-glucanase (AGN13.1) from the biocontrol fungus Trichoderma harzianum. Appl
Environ Microbiol, 67:5833-5839.
Altomare, C., Norvell, W. A., Bjorkman, T. & Harman, G. E. (1999a) Solubilization of phosphates and
micronutrients by the plant-growth-promoting and biocontrol fungus Trichoderma harzianum
Rifai 1295-22. Appl Environ Microb, 65:2926-2933.

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4.4.2.1. Size of the targeted markets
In most of the situations MBCAs are being developed with rather small, if not niche markets.
The total value of MBCAs sold worldwide amounted in 2008 to 620 Mio Euro (122 Mio Euro
in Europe) including products with insecticidal or fungicidal effects. This value can be
compared with the sales of chemical insecticides and fungicides amounting to a total of
21 000 Mio Euros.
MBCAs, with the exception of Bt products which can be used in larger crops such as grapes,
forestry or even cereals, are presently still used in speciality crops, greenhouses and
covered crops.
The size of these crops is not growing anymore or at a very reduced rate. The only optimistic
perspective is the intention to develop organic faster farming (objective 20% of the
production area in France in 2030) where MBCAs can find a good market.
Additionally the potential market is widely fragmented within a long list of crops such as
carrots, petersillium, onions, etc, usually referred to as “Minor crops”. These markets are so
small that even large chemical companies refrain from the investments that would cover the
needs and the manufacturers of MBCAs, due to the specificity of their products, are obliged
to invest and cover costs where scale economy can never be reached.
4.4.2.2. Cost of production
Contrary to the synthesis of chemicals, producing MBCAs requires a complicated and
extremely expensive process of production which can be divided into 4 phases:
- fermentation
- extraction
- purification
- formulation and packaging
All these phases are difficult and require relatively heavy costs.
a) fermentation
This first step has to be undertaken either with solid or with liquid phase technology.
Although the liquid phase fermentation is usually simple and cost effective, the
process is more risky because the produced spores are more fragile. In the contrary
using solid fermentation substrates will produce stronger, but it becomes more
difficult to increase the production volume.
b) extraction
Here again, there is a very strong difference between the MBCAs produced in liquid
or in solid fermenters.
In a liquid, the extraction will be rather easy by filtration, but the product will need to
be dried, which is a very long, energy-demanding and expensive process.
From a solid fermentation process, the extraction will be mechanical. Such a process
is rather harmful for the spores: It is again energy demanding and it is extremely
difficult to extract more that 60% of the spores from a substrate. In such a case the
productivity becomes rather poor.
c) purification
This step is very important to ensure the stability of the MBCAs produced. The
industrially produced MBCAs always contain impurities which, although biologically
inactive, may become critical over time, potentially creating risks of degradation,
inactivation etc.
In all situations the purification step requires a high level of sophistication and
expensive processes.

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d) Formulation and packaging
Formulation and packaging of MBCAs, due to their living state (and the requirement
that they remain alive for satisfactory effectiveness of the product), constitute an
extremely difficult step and in any case more expensive than the equivalent process
for chemicals. The choice of co-formulants, adjuvants and packaging material must
secure the quality of the MBCAs and its vitality. This is again a source of problems
and heavy costs.
Additionally to all the above mentioned hurtles, it has to be secured that no contamination
will occur, during the fermentation process naturally, but also during the extraction, the
purification, the formulation and the packaging. All the safety measures are very expensive
to carry out, but they are necessary in order to ensure the quality of the product brought to
the market
As a consequence of all these extra expenses and technical difficulties the MBCAs used for
this analysis were more than 4 times more expensive to produce than an equivalent
chemical pesticide (Table 24).
Table 24: Compared structure of the production costs for a microbial biocontrol
agent (MBCA) and a chemical insecticide (source IBMA).
Typical Insecticide MBCA Comments
Sales value 100 100
Type of production cost
Raw materials %* 8 29 40% lost material for
MBCA by solid
fermentation process
Packaging 1 2
Energy and miscellaneous 1 2
Manpower 5 9
Consumables 2 3
Amortisation 4 11
TOTAL 21 56
* costs are expressed as percent of the sales value of the commercial product
4.4.2.3. Cost of registration
It has been already mentioned that biological control agents suffer from a highly
unfavourable situation compared to chemical pesticides. The regulations for registration
have initially been set up to reduce the risks attached to molecules and the regulator is trying
to extrapolate these requirements for the registration of living organisms.
The estimated cost for registering a microbial biocontrol agent is currently lower than that for
a chemical pesticide (Table 25). However, the size of this investment is still very high for a
company in comparison with the market potential (
Table 26).This evaluation indicates that the introduction on the market of a MBCA is about 4
times less effective than its chemical equivalent.

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(Table 27). The gap between the two in terms of estimated profit is nearly 10-fold in favour
of the chemical industry.
Table 27: Compared margin structure estimates for the production and sales of a microbial
biocontrol agent (MBCA) and a chemical pesticide (source IBMA)
%*
Chemical
pesticide
MBCA
Sales value at plateau level 100 100
Costs of production 21 56
Gross margin 79 44
Cost of sales 21 15
Cost of research 8 12
Cost of administration 4 3
Earnings before investments
taxes and amortisation (EBITA)
46 14
Profit after taxes, provisions
and amortisation
18 ou 10? 2
* costs and margins are expressed as percent of the sales value of the commercial product
4.4.2.5. Conclusion and outlook for industry
These data show clearly that the profitability of a biocontrol business is much less attractive
than that of chemical pesticides and may explain why the large chemical companies decided
in the 90’s to retreat from this business. Although these companies show presently some
new signs of interest, they seem to remain basically reluctant to re-enter despite the new
attractiveness of a fast growing biocontrol market. Contrary to European and US-based
companies, several Japanese firms, such as Sumitomo chemicals or Mitsui appear to have
invested for a potential long term return. Taking advantage of the divestment by the chemical
majors, they have been able to acquire a good business basis at very attractive conditions.
This should enable them to consider optimistically the future development of the biocontrol
industry and its positive trend.
The smaller companies which have invested in this business and try to overcome their
financial problems have only two alternatives:
- Either develop, often at lost, into larger markets (grapevine, field crops etc), if they
can. In order to sustain these efforts, they will need a strong support from venture
capital companies;
- or enter into venture agreements with other manufacturers/suppliers, in order to build
up a product portfolio which will make them successful in the future.
4.4.3. Socio-economic aspects: market analysis and outlook
With estimated sales amounting to only 200 Mio€ in Europe in 2008, the market for
biological control agents appears to be extremely small compared with the 7 000 Mio€
turnover achieved with chemical pesticides. However, very important efforts have been
undertaken for the development of biocontrol agents. The OECD estimated that 5 000 Mio$
have been spent worldwide in public research for biocontrol during the last 40 years. This
amounts to a yearly average of 500Mio$, not far from the 600 Mio$ spent yearly in research
by the agrochemical industry, but with a comparatively poor result!
In the Conference on biological control organised in 2003 by IBMA in Béziers, France, the
major stakeholders (farmers, retailers, distributors, regulators etc.) have provided a list of
gaps considered to play a role in preventing wide adoption of biocontrol products. This list

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was meant to cover all potential explanations, but provided neither figures nor priority
ranking, making it difficult to prioritize actions for improvement. It was however a general
opinion that the complicated and costly system of registration was the major reason of the
problem. As a result, important efforts have been undertaken to convince the regulators to
adopt more facilitating procedures for the registration of biologicals. These efforts were not
without effect and the newly adopted “Pesticides package” makes it easier, under certain
conditions, to register biologicals. In the meantime, several EU member states have adopted
easier registrations tracks, such as the Biopesticides Scheme in the UK,.for example.
In reality, the unique assumption that the current regulations in Europe significantly hamper
the development and the use of biologicals does not seem to be proven by the facts. During
a very long period, the biologicals were not subject to registration and very few products
were brought successfully to the market. At the same time countries such as the USA, New
Zealand or Japan have adopted very liberal registration procedures, but the sales of
biologicals remain marginal.
In the frame of ENDURE, it has been therefore decided to get a detailed and quantified idea
on the gaps which, in Europe, restrain the adoption of biologicals, especially at the users and
commercial levels. In order to achieve this objective, a Pan-Europa survey was undertaken
from 2007 until 2008, with the assistance of the public opinion organisation Agridata.
4.4.3.1. Methodological approach: survey of European farmers
Since no validated data were available about the real market and the use of biological
control agents in Europe, it has been necessary to build up a form of electronic map of the
European agriculture and of the distribution of the potential users.
A survey was carried out to evaluate the size of the biocontrol market in Europe and to
identify key factors that could influence its future evolution. This study included four main
steps:
- Localisation of the main crops and cropping systems.
Using the data from EUROSTAT and national statistics a model of European agriculture
was constructed.
- Randomised sampling of farmers and retailers.
The model was used for the selection of 12 production systems (

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Table 28) located on 25 sites in 9 countries (Table 29) where 2000 farmers and 21
retailers were identified.
- The selected sample was contacted by phone directly and a questionnaire (Table 30)
was sent to those who agreed to participate in the survey. A total of 675 full responses
were obtained and analysed.
- Complementary survey.
In order to validate the process, more specific data was collected in a survey concerning
the biological control of wood diseases of grapevine in France

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4.4.3.2. Survey Results: The estimated market of biocontrol in Europe
The questionnaire made it possible to estimate the total biological market in ha and in value
(Figure 22) and its partition among different crops (Figure 23).
These data confirm that in 2008, the main use of biologicals was in protected crops, followed
by grapevine and fruit production. Nearly 40% of the estimated biocontrol market consisted
in sales of beneficial insects, compared to 25% for microorganisms and 21% for
semiochemicals (Figure 22).
52 Moi€ 12 Mio€
79 Mio€
18 Mio€
43 Mio€
Beneficial insects
Beneficial nematods
Microbial biocontrol agents
Semiochemicals
Natural substances
Total estimated EU sales of biocontrol products = 204 Mio€ in 2008
Figure 22: Estimated sales of biocontrol products in Europe in 2008 (in Million €). The
estimates were obtained by extrapolating use patterns in a representative sample
of EU farmers.
0
10
20
30
40
50
60
70
80
%oftotalProtected
crops
Field
vegetables
G
rapes
Fruits
Field
crops
G
ardens,ornam
entals
%of supply
%of acreage
Figure 23: Estimated distribution of biocontrol use among types of crops in 2008 in Europe

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4.4.3.3. Survey results: Factors of development of biocontrol
The exploitation of the questionnaires was somewhat difficult due to the large variety of
farmers and situations. Additionally, several open ended questions were introduced to collect
opinions on possible additional gaps and opportunities which were not mentioned in the
form.
Qualitative analysis:
In a first step, the analysis of the responses led to the identification of 12 factors deemed to
have a significant influence on the future development of biological control
9 factors with a positive influence:
o Ability of manufacturers to invest in R&D
o Financial strength of manufacturers
o Direct involvement of leading distributors
o Pull from the fresh food wholesalers and from the food industry
o Demand from consumers and NGOs
o Incentives given to growers
o Education of advisors and growers
o Availability of Decision Support Systems (DSS)
o Regulatory obstacles to chemical pesticides
3 factors with a negative influence:
o Regulations not adapted to Biological control
o Discovery of novel effective and safe chemicals
o Development of new resistant crops
Quantitative analysis
In a second step, a quantitative analysis was conducted to estimate the influence of the
identified factors. For this, 320 contacts (50% of the sample) were requested to indicate
which of the 12 factors they considered as important in terms of their potential impact on the
evolution of future use of biological control agents. For those factors selected as important,
the respondants were asked to weigh the expected impact positively or negatively on a scale
from 0 to 20.
The data were used to compute for each of the 12 factors:
a) an Influence Index, calculated as the percentage of respondants who selected the
factor as important
b) a Weight Index, calculated as the average of the weights attributed to the factor by
those respondants who selected it as important
c) a Growth Index, combining the two other indices according to the following formula:
GI = (Influence Index)*(Weight Index)/10
This index represents the overall estimate of the influence of a factor on the future
use of biological control agents by European farmers.
The scores computed for each of the 12 factors are presented in Table 31. Among the
factors deemed to carry the most impact on future use of biological control by European
farmers the action by far the most cited was the establishement of incentives for farmers
(factor D).

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Table 31: Impact of twelve factors on the future use of biocontrol agents by European
farmers according to a survey of 320 farmers
Factors
Influence
Index (%)*
Weight Index*
(scale from
-20 to +20)
Growth
Index*
Rank of
positive
influence
A
Registration for biological control
products remains as present
12 - 15 - 18.0
B Involvement of distribution 65 8 52.0 4
C Size / strength of the manufacturers 55 12 66.0 3
D Incentives to growers 87 18 156.6 1
E Education of advisors and growers 27 8 21.6 5
F Decision Support Systems available 12 7 7.2 9
G
Pull from wholesalers and food
industry
43 16 66.8 2
H Stringent registration of chemicals 16 14 22.4 6
I New safe chemical pesticides 42 - 12 - 3.0
J Progress in R&D of Biocontrol 8 14 11.2 8
K New resistant varieties 16 - 4 - 6.4
L Pull from Consumers 67 2 13,4 7
* see main text above for the specific definition of the indices
The second most important factors based on the Growth Index (G, C and B in Table 31)
were linked to the influence of key economic actors (the wholesalers, the food industry, the
distributors and manufacturers of biocontrol products).
The factors with the lowest scores were those related to scientific innovation (factors K, I, J).
Interestingly, both factors linked to regulatory aspects (factors H and A) also had a relatively
low Growth Index. The registration requirements are obviously more a concern for the
industry than for the users of the plant protection products.
Surprisingly, the efficacy and the price of the biologicals, usually considered as two critical
factors, were not mentioned as real constraints. This may be due to two reasons:
(1) It is anticipated that only “effective” solutions will be registered in the EU, showing the
high confidence of the farmers and the retailers in the registration systems
(2) The selling price of the new solutions (biological control products) will necessarily cope
with the current price levels. Too highly priced, the new solutions will simply be ignored.
4.4.3.4. Conclusions
The gaps and the opportunities for the development of biological crop protection products
are extremely relative to people concerned. While the industry, due to the heavy factor
time/cost to the market, considers the regulation requirements as a major obstacle, the users
and the retailers are much more influenced by the pull and push actions exercised at the
market level. Somewhat disappointing is the relative low concern about the technical
progress offered by the biological solutions.

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5. Conclusions and perspectives for future R&D projects
5.1.Improving classical and augmentative biological control
The review of published scientific literature on the biological control of selected pests and
diseases has lead to the identification of clear knowledge gaps highlighted in previous
chapters. Further bottlenecks were revealed by seeking the possible reasons for the striking
discrepancy between the rich inventory of potential biocontrol agents described by scientists
and a very small number of commercial products on the market.
To complement these analyses, consultations of experts (scientists, extension specialists
and representatives of the Biocontrol industry) were organised at the occasion of scientific
meetings of three Working Groups of IOBC-wprs1
.
- Working Group "Integrated Control of Plant Pathogens": meeting on "Molecular Tools for
Understanding and Improving Biocontrol" in Interlaken (Switzerland) September 9-12,
2008. (attended by P. Nicot and B. Blum – discussion session about the outlook on
biocontrol against plant diseases)
- Working Group “Multitrophic Interactions in Soil” meeting in Uppsala (Sweden), 10-13
June 2009. (attended by C. Alabouvette and C. Steinberg – roundtable about the outlook
on biocontrol of soilborne pests and diseases)
- Working Group "Insect Pathogens and Insect Parasitic Nematodes": meeting on "Future
Research and Development in the Use of Microbial Agents and Nematodes for Biological
Insect Control" in Pamplona (Spain), 22-25 June, 2009. (attended by C. Alabouvette –
his plenary presentations about the outlook on biocontrol of diseases and pests is
provided in Appendix 20)
These consultations were further complemented by discussions at the occasion of various
meetings of RA 4.3 participating partners to identify the most prominent issues that could be
tackled by future research and development activities. The key elements are organised
below in three categories, based on their relevance to the concern of the research
community, development or industry.
5.1.1. Key research issues
Five key issues have been identified in term of research needs:
 Devise better strategies for the screening of biocontrol agents. The demand for new
biocontrol agents is already high. It is expected to increase sharply in the EU, with
the ongoing reduction of available chemical pesticides and the need for new non-
chemical plant protection tools to comply with Directive 2009/128/EC (see chapter
4.3.1). Current methods need to be improved both in terms of logistics (high
throughput to allow rapid mass screening of large numbers of candidates) and in
terms of the pertinence of criteria for efficacy, production and commercialization. This
topic is currently being tackled within ENDURE RA 4.3 for microbial biocontrol agents
against diseases and the results will be presented as Deliverable 4.9.
 Improve knowledge on efficacy-related issues. The criteria traditionally used to
asses the efficacy of biological control methods may be misleading because
contrarily to conventional pesticides, biocontrol does not intend to eradicate pests
and diseases but, rather, to install a biological balance which will enable the plants to
1
International Organisation for Biological and integrated Control of noxious animals and plants – West
Palaearctic Regional Section

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grow more healthily. However the consistency of field efficacy remains one of the
constraints for the large scale use of biological control of plant diseases. Despite
much recent progress, research efforts are still necessary for (1) a better
understanding of key parameters of field efficacy in relation to the type of biocontrol
agent and their modes of action and (2) implementing the most promising methods
for efficacy improvement. Promising avenues of research are to be sought both in
terms of exploiting the biological properties of the biocontrol agents and enhancing
their effectiveness through formulation of the products. Results obtained on these
topics should provide key information both for the design of optimised production and
application strategies, but also for improving the screening process of future
biocontrol agents as mentioned in the point above.
 Promote multidisciplinary approaches to integrate better biocontrol with IPM and
other production issues. Based on passed published experience, it is clear that
levels of protection provided by a single biocontrol agent alone will seldom be
sufficient, especially when faced with field conditions unfavourable to their
effectiveness or with very high inoculum pressures of a pest or plant pathogen. More
emphasis will need to be placed on the compatibility of biocontrol agents with the
implementation of IPM, preferably in a systemic approach of integrated production.
Among the many possible interactions to be considered, compatibility and combined
used of biocontrol and plants genetically modified for improved resistance to pest or
plant diseases should not be overlooked.
 Develop adapted delivery technologies. Much progress has been made in packaging
technology and delivery for macrobial biocontrol agents (e.g. beneficial arthropods).
In contrast, treatments with microbial biocontrol agents (against pests or diseases)
still rely on sprayers developed for the application of pesticides. Research is needed
to provide growers with low pressure spraying equipment to preserve the viability of
the microbials. Technological improvements are also needed for optimal coverage of
the target plant surfaces to be protected by the biocontrol agents.
 Safeguard the durability of biocontrol. Certain pests and pathogens are known for
their capacity to develop resistance to chemical pesticides or to overcome varietal
resistance. The durability of biological control has often been assumed to be higher
than that of chemical control, but several examples of resistance of pests have
already been reported. Much less is known about plant pathogens, probably in part
because biological control against diseases is still very rare. Significant research
efforts are needed to anticipate the potential hurtles in this domain and integrate
durability concerns both in the screening of new biocontrol agents and in the careful
management of their use once they become commercially available.
5.1.2. Issues for development
Three key issues have been identified in terms of development. They are directly related to
improving the efficacy of crop protection but also to acceptability of biocontrol by farmers.
 Training of advisers and farmers. Compared to chemical control, the implementation
of biological control presents an additional level of technical complexity when the
"active substance" is a living organism or microorganism, whose liveliness and
development on the target crop underpins the effectiveness of the protection. In
many situations, achievement of successful biocontrol of pests has been linked to an
active role of advisers in accompanying the farmers, at least during their initial phase
of adoption and implementation. The success of large scale use of biological control
in the future will require stepping up the technical training of farmers and of advisors.
Such action will also positively influence the adoption issues mentioned below.
 Development and dissemination of Decision Support Systems (DSS). Growers
routinely make decisions that take into account multiple constraints (both technical

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and economic) of their activity. However, the complexity of biocontrol and its
necessary integration in a systems approach of crop protection and crop production
make DSS more and more indispensible, including in their function as easily
consultable repositories of knowledge on available choices.
 Establishment of demonstration schemes and development of farmers' networks.
This action is needed to stimulate the dissemination of information to and among
farmers, but also to facilitate exchange between the end users of biocontrol and the
other actors of research, development and commercialization of the products.
Breaking up regional and national barriers and including a European dimension to
such networks is desirable for optimal efficacy of multisite experimental trials.
5.1.3. Industrial issues
 Quality control. Ongoing efforts by the manufacturers of biological control agents to
guarantee the quality of their products need to be stepped up. The definition of tests
and their routine implementation is crucial to ensure reliable effectiveness and
maintain confidence of farmers for biocontrol. Whenever possible, such tests should
include not only an evaluation of viability of the biocontrol agent but also an
evaluation of physiological parameters related to its efficacy, based on knowledge of
its modes of action.
 Improve distribution systems.
5.2.Conservation Biological Control
The meta-review of Conservation Biological Control (CBC) research on invertebrate pests
has analysed 90 review papers published from 1989 to 2009 that address a very significant
body of primary literature. We identified and analysed 221 reports of research into CBC in
the review papers. Few of the review papers were published before 1998.
Europe has made strong contributions to both the primary literature and the review literature
concerning CBC and, together with the USA, has a leading position in this field of research.
Countries in the European Union were involved with 63% of all reports of CBC research
analysed and half of the institutions contributing to authorship of the reviews were European.
The main elements of research efforts in Europe were the same as those found in all the
papers reviewed but with a greater emphasis on arable crops.
Arable cropping systems with annual or biennial crops dominated the reported research, as
is appropriate to reflect the land area that they cover and their status as dietary staples. Field
vegetables, maize, vines and orchards, all the subjects of ENDURE Case Studies, also were
well represented. However, little CBC research on maize was reported in Europe and none
on vines and there was only a single report of CBC research in glasshouse vegetables. This
highlights a need for primary research on CBC in Europe to extend to the full range of
significant crops, for all research results to be published and for more crops to be included in
reviews.
Our analysis identified ten categories and 48 sub-categories of CBC practices or techniques
and revealed a clear emphasis in the literature on research into the management of
resources and refugia to support and promote natural enemies. In addition, investigations
into how these might best be managed at a landscape scale comprised 19% of reports in the
reviews. The great majority of CBC research that was reported was conducted at the field
scale. No studies were exclusively laboratory-based. Modelling was used in 5% of the
research reported and was a field in which European institutions were particularly well
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The most commonly cited target pests for CBC were Hemiptera (mostly aphids) and
Lepidoptera, in keeping with their status as agricultural pests. Predators and parasitoids
were the most commonly discussed natural enemies of Hemiptera and Lepidoptera,
respectively.
The great majority of reports provided evidence for the degree of success of CBC
techniques, especially in promoting natural enemies. The provision of refugia and resources
and landscape management were not only the topics that received most attention but also
the CBC techniques for which the benefits were best demonstrated. Evidence of success
was particularly strong in field vegetables, vines and arable crops. Evidence of improved
pest control was less often provided and usually weaker, indicating an important gap in
research.
 Considerably less research has been directed towards CBC strategies that optimise
the impact of naturally-occurring populations of weed natural enemies than has been
done for invertebrate pests. The two groups of biological control agents that have
received most attention in weed CBC research are deleterious rhizobacteria and
granivorous carabids. The techniques with most potential for weed CBC appear to be
the management of crop residues by conservation tillage and by manipulation of crop
rotations, and management of habitats (refugia and resources) for invertebrates.
Management of crop residues could be used for CBC strategies to encourage both
strains of naturally-occurring deleterious rhizobacteria that are specifically harmful to
weeds and granivorous carabids that prey on weed seeds. Weed seed predation can
be limited by a shortage of suitable habitats and refugia for herbivores and seed
predation is greater in complex landscapes than in simple ones.
Major needs for further research identified by the review literature:
 ‘Landscape-scale interactions’ was the subject most frequently identified as a priority
for further research. Studies of the appropriate spatial scale for landscape
management for CBC and studies of the movement of natural enemies within the
landscape are needed.
 The comparative benefits of plants and habitats to natural enemies, their
management and their role as sources or sinks for natural enemies, and their relative
value to pests and to beneficial organisms were considered priorities for further
study.
 Community ecology, autecology and behavioural ecology were identified as high
priorities for further research, especially: the impact of natural enemy diversity,
trophic interactions and community dynamics on CBC; the study of traits and
population dynamics of natural enemies and their responses to habitats; the
manipulation of natural enemy behaviour (e.g. by exploiting chemical ecology, push-
pull, mixed cropping).
 The impact of increased biodiversity on CBC was frequently stated as a priority for
future research and was the subject of only 4% of reports of past research.
 Further study of spatial and temporal factors affecting CBC was recommended by 43
and 13 of the 90 review papers, respectively. Large scale and long term studies are
needed.
 Impact assessment: there is a need for more studies that assess the effect of CBC
practices on pest control. Most reports of CBC included an assessment of the
benefits to natural enemies but fewer than half also assessed the effect on pest
control. This probably reflects the difficulties inherent in proving a link between
natural enemy promotion and the depression of pest populations. The complexity of
trophic relationships and the scale over which they operate has practical implications
for the scale and complexity of experimental design. However, a proper assessment
of the impact of CBC is needed. It should focus on testing the effectiveness of CBC
in relation to pest control, reduction in pesticide use, improved crop yield and cost-

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benefit analysis. The impact in CBC of different natural enemy species should also
be assessed.
 Modelling was singled out by some of the reviews as a priority for further use in CBC
research. Provided that suitable parameters are available or can be estimated and
that predictions can be tested in existing landscapes, modelling could provide means
to tackle questions where scale and complexity make manipulative field
experimentation difficult.
 Several reviews advocated that non-arthropod natural enemies, particularly
entomopathogens, were worthy of more study for CBC.
 More research effort should be applied to the integration of CBC into IPM.
 The socio-economic drivers of the uptake of CBC by farmers were mentioned as a
priority for further research by a minority of these science-based reviews.
Research gaps on CBC of weeds include:
 In-depth research on the ecology of relationships between deleterious rhizobacteria
and plants and on the mechanisms of action against weeds.
 Design of crop rotations to optimise the development of specific strains of deleterious
rhizobacteria for weed suppression.
 A comprehensive study of the ecology of predation of weed seed by invertebrates
and vertebrates and its impact on weed populations.
 Research on manipulation of the soil environment to encourage predators of weed
seed, e.g. by conservation tillage systems.
 Demonstration of the extent that landscape diversification benefits carabid
populations within cropped land and assessment of the impact on weed control.
 Rigorous evaluation of the effectiveness of weed biological control projects and the
reasons for success or failure
Other challenges to the implementation of CBC discussed in the literature
Other challenges to implementation of CBC in agricultural practice that were cited included:
 A lack of interdisciplinary research was seen to hamper scientific advancement in
support of CBC.
 High research and development costs, particularly in relation to large landscape-
scale or long term studies.
 Insufficient knowledge transfer (including a shortage of taxonomic expertise for
natural enemy identification).
 Farmer reluctance due to: risk perception, perception that CBC is complex compared
to chemical control, transitional costs, cultural conservatism.
 The difficulty of designing policy to promote large-scale landscape changes that
would be implemented by individual farmers.
6. Technical annexes

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Appendix 5 continued.
CBC practice and
techniques group
specific CBC
practice or
technique
Number of times review papers referred to different practices and reported the effect on natural enemies or pests total
number
of times
practice
referred
to
evidence for effect on abundance or fitness of NE evidence for effect on pest control
good
evidence
for a
strong
decrease
some
evidence
for a
decrease
no
consistent
evidence
for an
increase or
decrease
some
evidence
for an
increase
good
evidence
for a
strong
increase
unspecified good
evidence
for a
strong
decrease
some
evidence
for a
decrease
no
consistent
evidence
for an
increase or
decrease
some
evidence
for an
increase
good
evidence
for a
strong
increase
unspecified
Increased biodiversity of NE
various 1 1 1
unspecified 1 2 2 1 3
All 2 2 2 1 1 4
Various alternative prey 1 1 1
various 2 1 1 2 3
All 3 1 1 3 4
Unspecified 1 1 3 1 4 5
All CBC practices 2 10 110 80 19 20 63 21 117 221

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6.6. Appendix 6. Representation of European countries and institutions in the authorship of the
reviews that were the source literature for this meta-review
Country
where
institutions
were
located
Number of
institutions
Names of institutions Number of
times
institutions
represented
in
authorship
Austria 2 Federal Office and Research Centre of Agriculture, Vienna 1
Ludwig Boltzmann-Institute for Biological Agriculture and Applied Ecology,
Vienna
1
Belgium 1 Université Catholique de Louvain 1
Denmark 1 University of Copenhagen 2
Finland 1 University of Helsinki 1
France 2 CIRAD Montpellier 1
INRA Montpellier 1
Germany 2 Federal Biological Research Centre of Agriculture and Forestry (BBA),
Kleinmachnow
1
Georg-August University, Göttingen 5
Hungary 2 Plant Protection Institute, Hungarian Academy of Sciences, Budapest 1
Vas County Plant Protection and Soil Conservation Service, Tanakajd 1
Italy 2 Turin University 1
Netherlands 5 Alterra, Wageningen University and Research Centre 2
Applied Plant Research (PPO), Wageningen University and Research Centre 1
Netherlands Institute of Ecology (NIOO - KNAW) 1
Plant Research International, Wageningen University and Research Centre 1
Wageningen University 5
Spain 1 University of Castilla-La Mancha 1
Sweden 1 Swedish University of Agricultural Sciences, Uppsala 2
Switzerland 5 Agroscope FAL Reckenholz 2
CABI Bioscience, Delémont 1
FiBL (Research Institute of Organic Agriculture) 2
University of Berne 1
University of Zurich 1
UK 24 ADAS UK Ltd., Wolverhampton 1
British Trust for Ornithology, Thetford 1
Cardiff University 4
Centre for Ecology and Hydrology, Dorchester 1
Edinburgh University 1
Horticulture Research International, Wellesbourne 3
IACR Long Ashton Research Station, Bristol 1
Lancaster University 1
Marshall Agroecology Limited 1
NERC Centre for Ecology and Hydrology, Monks Wood 1
NERC Centre for Population Biology, Imperial College London, Silwood Park 1
Rothamsted Research, Harpenden 5
Royal Society for the Protection of Birds, Edinburgh 1
The Game Conservancy Trust, Fordingbridge 5
The University of Kent, Wye Campus 1
University of Stirling 1
University of Birmingham 1
University of Bristol 1
University of East Anglia 1
University of Newcastle upon Times 1
University of Oxford 1
University of Plymouth 1
University of Reading 2
University of Sussex 1
All 75

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6.7. Appendix 7. Representation of non-European countries and institutions in the authorship of
the reviews that were the source literature for this meta-review
Country
where
institutions
were located
Number of
institutions
Names of institutions Total number
of times
institutions
represented in
authorship
Australia 5 CSIRO Entomology, Queensland 2
Charles Sturt University, Orange, New South Wales 5
La Trobe University, Melbourne 1
University of Queensland 1
University of Sydney 6
Canada 1 Laval University, Quebec 1
Indonesia 1 International Centre for Research in Agroforestry, Bogor 1
Israel 1 Hebrew University of Jerusalem 1
Japan 1 Institute of Biological Control 1
Kenya 2 ICIPE (International Centre of Insect Physiology and Ecology) 2
Tropical Soil Biology and Fertility Institute of CIAT, Nairobi 1
México 1 Universidad Nacional Autónoma de México 1
New Zealand 1 Lincoln University, Canterbury 18
USA 37 Clemson University 1
Cornell University, Ithaca, New York 1
Iowa State University 1
Lousiana State University 2
Miami University, Oxford, Ohio 1
Michigan State University 7
Montana State University 2
Nature Mark, Boise, Idaho 1
New York State Agricultural Experimental Station 1
North Carolina State University 3
Ohio State University 3
Oregon State University 2
Pennsylvania State University 1
Rutgers University, New Brunswick 1
Santa Clara University, CA 1
South Central Research and Extension Center, Clay Center; Nebraska 1
Southern Illinois University 1
USDA Appalachian Fruit Research Station 1
USDA Forest Service, Center for Semiarid Forestry 1
USDA Plant Science and Water Conservation Research Laboratory,
Stillwater
1
USDA Southeastern Fruit and Tree Nut Research Laboratory, Georgia 1
USDA Western Cotton Research Laboratory, Phoenix, Arizona 2
University of Arkansas 1
University of California, Berkeley 5
University of California, Davis 2
University of California, Santa Cruz 1
University of Florida 2
University of Idaho 1
University of Maryland 5
University of Massachusetts 2
University of Minnesota 6
University of Missouri-Columbia 1
University of Nebraska, Lincoln 2
University of Tennessee 1
University of Wisconsin 2
Utah State University 1
Washington State University 3
All 112

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6.9. Appendix 9. Categorization of challenges to implementation of CBC identified by authors of review papers
Challenge
category
Challenge sub-category: factors that impede progress toward the implementation of CBC (long description)
Short description of challenge sub-
category
Scientific
practice
Division of ecological, agronomic and socio-economic research amongst different disciplines and sub-disciplines hampers scientific advancement Lack of interdisciplinary research
R&D costs Experiments needed for CBC development may be costly, especially if they address landscape scales and long time periods Cost of large scale CBC experiments
Costs associated with registration of semiochemicals for field use are very high Cost of registration of semiochemicals
The difficulty in commodifying CBC makes it difficult to recoup costs of R&D and inhibits research investment by commercial companies Lack of financial return for R&D
Knowledge
transfer
Better knowledge transfer methods are needed to enable extension services to communicate CBC methods and skills Better knowledge transfer methods
Local ecological variation influences the success of CBC techniques and the knowledge necessary to support them Local ecological variation
Lack of taxonomic expertise to identify pests and NE's and lack of accessible yet authoritative identification guides or techniques Lack of taxonomic expertise
Socio-
economic
Perception of risk associated with CBC, lack of consistent evidence for success of CBC Perceived risk relating to CBC
Perceived complexity of implementation of CBC in comparison to conventional chemical-based control Perceived complexity of CBC
Cultural impediments to change in agricultural practice Cultural impediments to change
Cost of implementing CBC measures, including transitional costs Cost of implementing CBC
Policy Design of policy instruments promoting large-scale landscape changes to support CBC but depending upon actions on individual farms Landscape-scale implementation of CBC
Complexity of agri-environment schemes and their multiple functions Complexity of agri-environment schemes
Increasing both crop production and habitat diversity for CBC Achieving productivity with biodiversity
Abbreviations used in Appendix 9 CBC: conservation biological control NE: natural enemy R&D: research and development

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6.15. Appendix 15. Primary literature (2007-2009) on biological control against Fusarium oxysporum
Abo-Elyousr, K. A. M. and H. M. Mohamed (2009). "Biological Control of Fusarium Wilt in Tomato by Plant Growth-Promoting Yeasts and Rhizobacteria." Plant Pathology Journal 25(2):
199-204.
Three plant growth-promoting yeasts and two rhizobacteria were tested for controlling tomato wilt caused by Fusarium oxysporum L sp. lycopersici under greenhouse and field
conditions. Under greenhouse and field conditions, all treatments were significantly reduced disease severity of tomato wilt relative to the infected control. The highest disease
reductions in pots (75.0, 67.4%) and field (52.5, 42.4%) were achieved by Azospirillum brasilense and Bacillus subtilis compared to infected control. Under field condition all treatments
produced the highest tomato yield compared to the control plants inoculated with the pathogen
.
Al-Jedabi, A. A. (2009). "Biological control of Fusarium root-rot of sorghum." Research Journal of Agriculture and Biological Sciences 5(4): 465-473.
several crops including sorghum that result in low grain yield. All antagonists showed inhibition of mycelial growth of F. oxysporum and the maximum inhibition was recorded when
Bacillus subtilis as biocontrol agent (67.7%). The in vitro root colonization study demonstrated that after four days of germination, the cell counts obtained from the roots have
increased and the maximum count is achieved by B. subtilis (16.9*105 cfu/cm root). The greenhouse pot experiment demonstrated that T. viride and B. subtilis resulted in more than
80% suppression of root rot. The reduction in fresh weight of roots amounted to 93.6% in the control treatment inoculated with F. oxysporum alone, whereas 71.1% reduction in fresh
root weight was recorded for the treatments inoculated with both the pathogen and B. subtilis; 66.8% reduction in fresh root weight was recorded for the treatments inoculated with
both the pathogen and T. harzianum. Root dry weight of the control treatment inoculated with only F. oxysporum decreased by 94.5% in relation to the non-inoculated control. Among
the potential biological control agents in this study, B. cereus resulted in 42.3 reduction in root dry weight compared to the 94.5% reduction recorded for the control inoculated with F.
oxysporum alone. 100% of the roots from the control treatment (F. oxysporum only) rendered growth of F. oxysporum compared to an incidence ranging from 20 to 55% for plants
treated with B. subtilis, B. lecheniformis, B. cereus, T. harzianum and T. viride. Both chlorophyll fractions increased when treated with antagonist and the maximum enhancement was
recorded when Bacillus subtilis used as antagonist compared with that of control. The maximum values of the carbohydrate components were recorded when Bacillus subtilis used as
antagonist relative to those of control.
Amini, J. (2009). "Induced Resistance in Tomato Plants Against Fusarium Wilt Invoked by Nonpathogenic Fusarium, Chitosan and Bion." Plant Pathology Journal 25(3): 256-262.
The potential of nonpathogenic Fusarium oxysporum strain Avr5, either alone or in combination with chitosan and Bion, for inducing defense reaction in tomato plants inoculated with
E oxyysporum f. sp lycopersici, was studied in vitro and glasshouse conditions. Application Bion at concentration of 5, 50, 100 and 500 mu g/ml, and the highest concentration of
chitosan reduced in vitro growth of the pathogen. Nonpathogenic F oxysporum Avr5 reduced the disease severity of Fusarium wilt of tomato in split plants, significantly. Bion and
chitosan applied on tomato seedlings at concentration 100 mu g a.i./plant; 15, 10 and 5 days before inoculation of pathogen. All treatments significantly reduced disease severity of
Fusarium wilt of tomato relative to the infected control. The biggest disease reduction and increasing tomato growth belong to combination of nonpathogenic Fusarium and Bion.
Growth rate of shoot and root markedly inhibited in tomato plants in response to tomato Fusarium wilt as compared with healthy control. These results suggest that reduction in
disease incidence and promotion in growth parameters in tomato plants inoculated with nonpathogenic Fusarium and sprayed with elicitors could be related to the synergistic and
cooperative effect between them, which lead to the induction and regulation of disease resistance. Combination of elicitors and nonpathogenic Fusarium synergistically inhibit the
growth of pathogen and provide the first experimental support to the hypothesis that such synergy can contribute to enhanced fungal resistance in tomato. This chemical could provide
a new approach for suppression of tomato Fusarium wilt, but its practical use needs further investigation.
Anand, R., S. Kulothungan, et al. (2009). "Assay of chitinase and beta-1,3 glucanase in Gossypium hirsutum seedlings by Trichoderma spp. against Fusarium oxysporum." International
Journal of Plant Sciences (Muzaffarnagar) 4(1): 255-258.
wilt in cotton. In this regard, the six species of Trichoderma, namely T. viride, T. virens [Gliocladium virens], T. hamatum, T. harzianum, T. koningii and T. reesi, were evaluated for its
biocontrol properties and induction of defence-related enzymes, namely chitinase and beta1-3-glucanase in 30 days old cotton (G. hirsutum) seedlings. Trichoderma spp. could
efficiently control the growth rate of F. oxysporum. In vitro assay of chitinase and beta-1,3-glucanase revealed the maximum production by T. harzianum (56 U/ml) and T. hamatum
(80 U/ml), respectively. It also produced appreciable quantities of defence enzymes. The maximum induction of chitinase and beta1-3-glucanase in plants was found to be 80 U/ml
when challenged with T. harzianum, in addition to the enhancement of defence mechanism in plants. Trichoderma spp. improved the germination rate of seedlings.

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Anitha, A. and M. Rebeeth (2009). "Self-fusion of Streptomyces griseus enhances chitinase production and biocontrol activity against Fusarium oxysporum f. sp. lycopersici." Biosciences,
Biotechnology Research Asia 6(1): 175-180.
Protoplasts were isolated from Streptomyces griseus (MTCC - *4734) strain using lysing enzymes and self-fusion of Streptomyces griseus protoplasts was carried out using 50%
polyethylene glycol (MW 1000, Sigma Chemicals Co., USA) in protoplast buffer. The regenerated 8 self fused Streptomyces griseus were studied detailed for chitinase production and
biocontrol activity. Parent strain (PSg) showed protein content of 2.7 mg/ml with chitinase activity of 120 IU/ml. High chitinase activity was measured in the culture filtrates of most of
the self-fusants (87%) than the parent. Among the fusants, the strain SFSg 5 produced protein content of 7.8 mg/ml, maximum chitinase activity of 283.3 IU/ml with a two-fold increase
as compared to the parent strain. All the self-fusants exhibited increased antagonistic activity against F. oxysporum f. sp. lycopersici than the parent. Maximum inhibition (82%, 80%) of
mycelial growth of F. oxysporum was recorded with fusant of SFSg 5, SFSg 1 as against 61.1% with PSg. The result implies that, the self-fused Streptomyces griseus resulted in
appreciable increase of chitinase production and biocontrol activity also the significance of the protoplast fusion technique, which could successfully be used to develop hybrid strains
also for commercial formulation.
Baysal, O., M. Calskan, et al. (2008). "An inhibitory effect of a new Bacillus subtilis strain (EU07) against Fusarium oxysporum f. sp. Radicis-lycopersici." PMPP Physiological and Molecular
Plant Pathology 73(1/3): 25-32.
destructive disease on tomato (Lycopersicon esculentum Mill.) transplant seedlings and the causal organism of crown and root rot of tomato plants growing in southern coast
greenhouses of Turkey. An isolate of Bacillus subtilis (EU07) identified by the 16s RNA region code gene was selected as the best antagonist and evaluated against FORL in vitro
studies. Strain EU07 at 106 CFU ml-1 was able to reduce disease incidence by 75%, when applied as an inoculant. It efficiently inhibited FORL compared to the control and QST 713
(AgraQuest, Davis, CA) whose inhibition ratio was only 52% in vivo. Random amplified polymorphic DNA analyses showed banding (genetic) differences between EU07 and QST 713
whereas there were no differences between DNAs of strains that have high homology to genes involved in the synthesis of antibiotics fengycin, bacillomycin and iturin when screened
by oligonucleotide primers designed based on sequence information obtained from the NCBI database. Furthermore, one specific fragment in the EU07 genome showed the highest
similarity to YrvN protein by 99% and AAA ATPase domain protein (72.2%) after amplifying oligonucleotide primers that are specific to the N-acyl-homoserine lactonase (HLS) gene
as a biocontrol activity marker. These results suggested an effect of EU07 on control FORL by YrvN protein as subunit of protease enzyme. Furthermore, this fragment associated
with HLS gene may be a potential molecular marker for selecting effective biological control agent belonging to Bacillus in order to control soilborne pathogens such as Fusarium,
suggesting impairment in FORL invasion by signaling in the plant rhizosphere.
Bernal-Vicente, A., M. Ros, et al. (2009). "Increased effectiveness of the Trichoderma harzianum isolate T-78 against Fusarium wilt on melon plants under nursery conditions." Journal of
the Science of Food and Agriculture 89(5): 827-833.
BACKGROUND: The use of isolates of the genus Trichoderma to control Fusarium wilt in melon plants is one of the most recent and effective alternatives to chemical treatments. In
this work we have studied the immobilization of the isolate Trichoderma harzianum T-78 on different carriers as an efficient method to control vascular Fusarium wilt of melon in
nurseries. Different formulations were developed: liquids (spore suspension, guar gum and carboxymethylcellulose) and solids (bentonite, vermiculite and wheat bran). RESULTS:
The introduction of F. oxysporum resulted in a significant decrease in seedling fresh weight. The treatments which gave a lesser reduction in weight and showing a greater biocontrol
effect were the liquid conidial suspension and the solid treatments with bentonite and superficial vermiculite. Microbiological analyses revealed that the conidial suspension and all the
solid treatments, except wheat bran, significantly decreased F. oxysporum populations. Of all the treatments assayed, bentonite produced the greatest decline in the F. oxysporum
population. CONCLUSIONS: The most effective treatments against Fusarium wilt on melon plants were the solid treatments bentonite and superficial vermiculite. These two
treatments gave the greatest plant weight, the lowest percentage of infected plants and the greatest T. harzianum population throughout the assay. (C) 2009 Society of Chemical
Industry
Boureghda, H. and Z. Bouznad (2009). "Biological control of Fusarium wilt of chickpea using isolates of Trichoderma atroviride, T. harzianum and T. longibrachiatum." Acta
Phytopathologica et Entomologica Hungarica 44(1): 25-38.
The efficiency of the antagonist species Trichoderma atroviride (strains Ta.3, Ta.7 and Ta.13), T. harzianum (Th.6, Th.12, Th.15, Th.16 and Th.18) and T. longibrachiatum (TL.1, TL.2,
TL.4, TL.5, TL.8, TL.9, TL.10, TL.11, TL.14 and TL17) against Fusarium wilt (caused by Fusarium oxysporum f.sp. ciceris) was compared using in vitro- and in vivo-based bioassay. A
significant decrease of both growth and conidia production of the pathogen was obtained compared to the control. The highest percentages of diameter colony reduction and conidial
production were obtained with Ta.13, causing 65.64% reduction in colony diameter (direct confrontation), 48.71% reduction in colony diameter (indirect confrontation), and a complete

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inhibition of conidial production. Once more in direct confrontation, T. atroviride overgrowth the pathogen colony and sporulate above. The seed treatment by Trichoderma spp.
isolates before sowing in a soil already infested by the pathogen led to a significant decrease of disease severity compared to the untreated control. The weakest index of disease
severity was obtained with Ta.13, which caused 83.92% reduction compared to the control. The most effective isolates in protecting chickpea seedlings against the disease were Ta.3,
Ta.7 and Ta.13 as well as Th.16. The reduction of disease severity was associated with an increase of the vegetal growth including the stem height as well as the plant fresh and dry
weights.
Casimiro Michel-Aceves, A., M. Antonio Otero-Sanchez, et al. (2009). "In vitro biocontrol of Fusarium subglutinans (Wollenweb. and Reinking) Nelson, Toussoun and Marasas and F.
oxysporum Schlecht., causal agents of "Witches' broom" of mango (Mangifera indica L.) by Trichoderma spp." Revista Mexicana de Fitopatologia 27(1): 18-26.
The antagonistic effect of native strains of Trichoderma spp. was evaluated in vitro against Fusarium oxysporum (Fo) and Fusarium subglutinans (Fs), causal agents of mango
"witches' broom". Ten strains of the antagonistic fungus were isolated, one of which was selected and identified to the species level (T. harzianum); this species showed the highest
percentage of antagonism inhibiting mycelial growth of Fo by 62.9% and 42.0% of Fs. In dual Cultures between Fo and/or Fs with the selected strains of Trichoderma, the time for the
first contact for Fo was between 3 and 4 days, and between 2 and 3 for Fs. The greatest intersection area (0.87 cm) was observed in T. lignorum against Fo, while the intersection
area in Fs with the native strain Thzn-2 was 0.85 cm. Native strains Thzn-2 and Thzcf-12, and the commercial one showed antagonism class 2, being able to stop growth of both plant
pathogens. Strain Thzn-2 is promising as an alternative for biocontrol of Fo and Fs; however, it is necessary to evaluate it under field conditions.
Chebotar, V. K., N. M. Makarova, et al. (2009). "Antifungal and phytostimulating characteristics of Bacillus subtilis Ch-13 rhizospheric strain, producer of bioprepations." Applied
Biochemistry and Microbiology 45(4): 419-423.
Bacillus subtilis Ch-13 industrial strain was shown to have a wide spectrum of antagonistic activities against different species of phytopathogenic fungi and bacteria. The B. subtilis Ch-
13 strain produces lytic enzymes; cyanide and other antifungal metabolites; stimulates plant growth, producing phytohormones-auxin derivatives. This strain by 2.5 times reduced the
quantity of tomato plants infected with phytopathogenic fungus Fusarium oxysporum during inoculation. Fungi abundance on roots with bacterial inoculation was 6.9 times less than in
the absence of inoculation. The application of detected antifungal metabolites as biochemical markers for the strain enables to control the stability of physiologic and biochemical
characteristics of the producer, and ensures a rapid quality assay of biopreparations with high performance liquid chromatography (HPLC).
Chen, L. and W. Chen (2009). "Genome shuffling enhanced antagonistic activity against Fusarium oxysporum f. sp. melonis and tolerance to chemical fungicides in Bacillus subtilis BS14."
Journal of Food, Agriculture & Environment 7(2): 856-860.
enhance antagonistic activity against Fusarium oxysporum f. sp. melonis (FOM) and tolerance to two chemical fungicides. Strain BS14 was identified as a strain of Bacillus subtilis by
the analysis of 16S rDNA sequences. A stable recombinant F35 was obtained after three rounds of shuffling. Antagonistic activity of recombinant F35 against FOM was increased by
34.52% and 65.48% compared to that of the parent strain HN8-7 with highest activity and another parent strain utilized, BS14. The tolerance to chemical fungicides was also
significantly improved (p0.05) compared to that of strain BS14. Reduction of FOM of 94% was observed by using recombinant F35, which was increased by 45% compared to that of
strain BS14 (p0.05) and no significant differences (p>0.05) compared to that of thiophanate methyl (MRL). Reduction of FOM of 100% was dramatically observed by using an
integrated treatment combining MRL (50% of usual dosage) with recombinant F35. Strain F35 with these improved traits would be a promising biocontrol agent in the control of FOM.
Here genome shuffling was proved to be a practical methodology for strain improvement of antagonistic microorganism Bacillus subtilis BS14 for enhancing antagonistic activity
against FOM and tolerance to chemical fungicides.
Clematis, F., M. L. Gullino, et al. (2009). "Antagonistic activity of microorganisms isolated from recycled soilless substrates against Fusarium crow rot." Protezione delle Colture(3): 29-33.
We report the results obtained in biological control trials against crown and root rot of tomato incited by Fusarium oxysporum f. sp. radicis lycopersici by using microorganisms isolated
from soilless cultivation systems that showed suppressiveness against this disease. Among the tested microorganisms belonging to fluorescent bacteria (32 isolates) and to fungi
belonging to Trichoderma (39 isolates) and Fusarium (38 isolated), 5 bacteria and 6 fungi showed a good activity against the pathogen. Such strains will be used in greenhouse trials,
under situations closer to the field, in order to evaluate their potential to be adopted under practical conditions.
Eden Paredes-Escalante, J., J. Armando Carrillo-Fasio, et al. (2009). "Antagonistic microorganismos for control of the fungal complex that cause wilt in chickpea (Cicer arietinum L.) in the
state of Sinaloa, Mexico." Revista Mexicana de Fitopatologia 27(1): 27-35.

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The antagonistic activity in vitro of microorganisms isolated from chickpea rhizosphere, was evaluated against Fusarium oxysporum, Sclerotium rolfsii, and Rhizoctonia solani, causal
agents of chickpea wilt. The native strains with the higher percentage of pathogen mycelial growth inhibition were selected and identified as Trichoderma lignorum (CIAD 06-540903),
T. harzianum (CIAD 05-550903), Bacillus subtilis (CIAD-940111), and Pseudomonas fluorescens (CIAD-990111). These strains and a commercial strain of T. harzianum (T-22) were
mixed with Glomus intraradices and their effectiveness to reduce chickpea wilt was compared against a chemical treatment (PCNB) and all absolute control in the field. The seed was
treated with the microorganisms before sowing and evaluations of disease severity were conducted each 15 days, while root colonization by the antagonistic microorganisms was
assessed 45 days after sowing. Colonization of T, harzianum CIAD 05-550903 + G. infraradices was 33 x 10(3) ufc/g fresh root-75% and B. subtilis + G. intraradices was 1.3 x 10(8)
Ufc/g fresh root-75%; while the combination P.fluorescens + G. intraradices was 1.4 x 10(7) Ufc/g fresh root-88%. These treatments also showed a reduction of disease severity in 64,
57, and 51%, respectively in comparison with the control.
El-Khallal, S. M. (2007). "Induction and modulation of resistance in tomato plants against Fusarium wilt disease by bioagent fungi (arbuscular mycorrhiza) and/or hormonal elicitors
(jasmonic acid & salicylic acid): 2 - changes in the antioxidant enzymes, phenolic compounds and pathogen related-proteins." Australian Journal of Basic and Applied Sciences 1(4):
717-732.
Induction of plant defense against pathogen attack is regulated by a complex network of different signals. In the present study interaction between hormonal signals [jasmonic acid
(JA) or salicylic acid (SA)] and bioagent [arbuscular mychorrhiza (AM) fungi] was used as new strategy to enhance tomato defense responses against wilt disease caused by
Fusarium oxysporum (Fo). Thus changes in various physiological defenses including antioxidant enzymes, phenolic compounds and pathogenesis related (PR) proteins were
investigated in leaves of tomato plants. Results appeared that production of reactive oxygen species (ROS), mainly H2O2 and O2 increasing the time of infection. Application with
bioagent AM fungi and/or hormonal elicitors (JA & SA) markedly decreased these levels, while LOX activity greatly increased as compared with infected control. SA - treated plants
had the highest MDA level but JA+AM fungi treated plants recorded the highest LOX activity. Infection by Fusarium oxysporm significantly increased activity of antioxidant enzymes
(SOD, APX and CAT) in tomato leaves at different stages of growth. The highest activity was recorded in leaves of AM fungi+JA-treated plants, while treatments with SA especially
when applied alone markedly decreased H2O2 scavenging enzymes (APX and CAT) and greatly increased SOD activity. Thus, imbalance between H2O2 - generation and
scavenging enzymes in leaves may reflect a defense mechanism in tomato or a pathogenicity strategy of the fungus. Levels of certain phenolic acids greatly changed in tomato leaves
in response to Fusarium oxysporum, AM fungi and hormonal elicitors. Benzoic and Galleic acids contents markedly decreased, however, contents of coumaric, cinnamic, chlorogenic
and ferulic acids increased in leaves of all treatments. Also, activity of lignification enzymes POX, PPX and PAL significantly increased in leaves of infected tomato plants. JA-treated
plants caused the highest POX and PPX activities, while SA-treated plants having the highest PAL activities. High accumulation of phenolic compounds and activity POX, PPX and
PAL in these plants may reflect a component of many defense signals activated by bioagent and hormonal inducers which leading to the activation of power defense system in tomato
against attack. Analysis of protein electrophoresis revealed that interaction between hormone signal (JA & SA) and bioagent AM fungi mediating the expression of the majority of
different PR-proteins leading to increasing defense mechanism against Fusarium oxysporum infection. Thus, induction of protein bands of molecular weights 35, 33, 32, 31 (PR-2,
beta-1, 3 glucanase), 30.5 and 27 (PR-3,-4, chitinase) in infected leaves indicated the important role which played in disease resistance. Finally, the new mechanism of the
combination strategy between bioagent and hormonal signals (either synergistically or antagonistically) played important roles for increasing various defense systems and altering
expression of defense genes which leading to different PR-proteins working together to increased resistance in tomato plants against wilt disease caused by Fusarium oxysporum. In
addition, results revealed that defense mechanism in plants treated with AM fungi and JA are more effective than AM fungi plus SA-treated plants.
Floch, G. l., J. Vallance, et al. (2009). "Combining the oomycete Pythium oligandrum with two other antagonistic fungi: root relationships and tomato grey mold biocontrol." Biological
Control 50(3): 288-298.
To reduce Pythium oligandrum biocontrol variability and improve its efficacy, experiments were performed by combining the oomycete with two other antagonistic fungi, Fusarium
dishes, Fo47 or T. harzianum hyphae destroyed P. oligandrum cells by antibiosis and mycoparasitism processes; in the rhizosphere of tomato plants (Lycopersicon esculentum), the
same antagonistic features were observed. However, in the rhizosphere, hyphae are frequently separated by a certain distance; this allows the coexistence and the persistence of the
three microorganisms on the root systems. When introduced in the rhizosphere, Fo47 and P. oligandrum were able to penetrate the root tissues with Fo47 limited to the epidermal and
upper layers of cortical cells while P. oligandrum colonized deeper tissue at a faster rate. The two antagonists were killed in few days within roots following elicited plant-defense
reactions. T. harzianum was not able to penetrate root tissues. Root colonization with either P.oligandrum alone or in combination with Fo47 and/or T. harzianum resulted in systemic
plant resistance which provided plant protection against Botrytis cinerea infection of leaves. The level of control and the expression of pathogenesis-related proteins (PR-proteins) in
leaves were similar whatever the antagonistic microbial treatment applied to roots.

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Gay, M. I. T., Anonymous, et al. (2009). Substrates containing a Trichoderma asperellum strain for biological control of Fusarium and Rhizoctonia, Universidad de Barcelona.
The strain of Trichoderma asperellum T34(2) CECT No. 20417 is useful for preparing substrates for biological control of vascular fusariose and death of plants caused by Rhizoctonia
solani. The substrates can be peats, composts (hardwood compost, pine bark compost, cork compost, sludge compost from sewage treatment plants, garden residues, etc.) or
formulations based on CPV-type compost (compost+peat+vermiculite). The fact that the substrates suppress both Fusarium oxysporum f. sp. lycopersici and Rhizoctonia solani
provides an advantage in comparison with other substrates known in prior art. Another advantage is that the use of methyl bromide, a highly harmful product for the environment, in
the control of vascular fusariose is avoided.
Huang, X., J. Luo, et al. (2009). "Isolation and bioactivity of endophytic fungi in Derris hancei." Journal of South China Agricultural University 30(2): 44-47.
Derris hancei Hemsl. The antagonism of endophytic fungi against fungal pathogens was tested in vitro. Penicillium sp. Q1, Rhizoctonia sp. S1, Phomopsis sp. N2, and Corticium sp.
F1 isolated from the caudex of D. hancei, and Penicillium sp. Q2 isolated from the leaf, inhibited the hyphal growth of Colletotrichum gloeosporioides Penz, Fusarium oxysporum f.
niveum (E. F. Smith) Snyber et Hansen, Rhizoctonia sp. S1 against Colletotrichum orbiculare Arx, and Phomopsis sp. N2 against Colletotrichum musae (Berk1 & Curt1) Arx1 on dual
culture with inhibition index II. It was reported that endophytic fungus in D.hancei could produced antibacterial substances in this paper. The culture filtrates of Penicillium sp. Q2
treated in 48 h after treatment possessed 100.00% of adjusted mortality against the 2nd larvae of Spodoptera litura by leaves disc feeding bioassays, and 75.10% against Lipaphis
erysimi Kaltenbach (apterous adult) by insect-soaking method, respectively, which showed that the activity of Penicillium sp. Q2 was higher than that of other endophytic fungi.
Jadeja, K. B. and D. M. Nandoliya (2008). "Integrated management of wilt of cumin (Cuminum cyminum L.)." Journal of Spices and Aromatic Crops 17(3): 223-229.
Four components of integrated management namely, soil solarization, crop rotation, chemicals and biocontrol agents were tested under field condition at Junagadh (Gujarat) for the
management of wilt of cumin (Cuminum cyminum) caused by Fusarium oxysporum f. sp. cumini. Growing of sorghum (Sorghum bicolor) or maize (Zea mays) during kharif season did
not reduce wilt incidence during the following rabi season. Soil solarization with 25 m LLDPE plastic cover for 15 days in summer proved most effective in reducing wilt incidence to
26.27% as against 44.90% in non-solarization and increasing yield to 396 kg ha-1 as against 286 kg ha-1 in non-solarized plots. Application of carbendazim granules @ 10 kg ha-1
one month after sowing or Trichoderma viride in organic carrier @ 62.5 kg ha-1 at sowing time were also effective. Integrating soil solarization followed by growing of sorghum in kharif
and application of either carbendazim granules @ 10 kg ha-1 one month after sowing or application of T.viride in organic carrier @ 62.5 kg ha-1 was effective for the management of
cumin wilt.
Kamilova, F., S. Validov, et al. (2009). Biological control of tomato foot and root rot caused by Fusarium oxysporum f.sp. radicis-lycopersici by Pseudomonas bacteria. Proceedings of the
Second International Symposium on Tomato Diseases, Kusadasi, Turkey, 8-12 October 2007.
Rhizobacteria are a natural and most suitable source for the isolation of potential microbiological control agents that can protect plants from soilborne pathogens and consequently
improve crop quality and yield. The beneficial effect of such bacteria on plant health depends in many cases on their ability to aggressively colonize the rhizosphere and compete with
the indigenous, including pathogenic, microflora for nutrients and niches on the plant root. Bacterial strains Pseudomonas chlororaphis PCL1391 and P. fluorescens WCS365 employ
antibiosis and induced systemic resistance, respectively, to control tomato foot and root rot (TFRR) caused by phytopathogenic fungus Fusarium oxysporum f.sp. radicis-lycopersici
(Forl). For the selection of biocontrol bacteria acting via the mechanism "competition for nutrients and niches" we have developed an enrichment method for enhanced tomato root tip
colonizers, starting from a crude mixture of rhizobacteria coated on the seed, using a sterile quartz sand/plant nutrient solution gnotobiotic system. As a result of this enrichment
procedure, and subsequent tests on competitive tomato root tip colonization, the strongly competitive biocontrol strains P. fluorescens PCL1751 and P. putida PCL1760 were isolated.
Both strains effectively suppress TFRR under soil and hydroponic cultivation conditions.
Kamilova, F., S. Validov, et al. (2009). "Biological control of tomato foot and root rot caused by Fusarium oxysporum f.sp. radicis-lycopersici by Pseudomonas bacteria." Acta
Horticulturae(808): 317-320.
isolation of potential microbiological control agents that can protect plants from soilborne pathogens and consequently improve crop quality and yield. The beneficial effect of such
bacteria on plant health depends in many cases on their ability to aggressively colonize the rhizosphere and compete with the indigenous, including pathogenic, microflora for nutrients
and niches on the plant root. Bacterial strains Pseudomonas chlororaphis PCL1391 and P. fluorescens WCS365 employ antibiosis and induced systemic resistance, respectively, to
control tomato foot and root rot (TFRR) caused by phytopathogenic fungus Fusarium oxysporum f.sp. radicis-lycopersici (Forl). For the selection of biocontrol bacteria acting via the
mechanism "competition for nutrients and niches" we have developed an enrichment method for enhanced tomato root tip colonizers, starting from a crude mixture of rhizobacteria
coated on the seed, using a sterile quartz sand/plant nutrient solution gnotobiotic system. As a result of this enrichment procedure, and subsequent tests on competitive tomato root tip

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colonization, the strongly competitive biocontrol strains P. fluorescens PCL1751 and P. putida PCL1760 were isolated. Both strains effectively suppress TFRR under soil and
hydroponic cultivation conditions.
Kannan, V. and R. Sureendar (2009). "Synergistic effect of beneficial rhizosphere microflora in biocontrol and plant growth promotion." Journal of Basic Microbiology 49(2): 158-164.
Biological systems are getting more relevance than chemical control of plant pathogens as they are not only eco-friendly and economic in approach but are also involved in improving
the soil consistency and maintenance of natural soil flora. Plant growth promoting rhizosphere microorganisms were isolated from three different tree rhizospheres using selective
culture media. Five microorganisms were selected from each rhizosphere soil based on their efficiency and screened for their ability to promote plant growth as a consortium. Each of
the developed consortium has a phosphate solubilizer, nitrogen fixer, growth hormone producer, heterotrophic member and an antagonist. The plant growth promoting ability of the
microbial members present in the consortium was observed by estimating the IAA production level and also by the nitrogenase activity of the nitrogen fixers. The biocontrol potentiality
of the consortium and the antagonist present in the consortium were checked by both dual plate assay and cross-streaking technique. Consortial treatments effected very good growth
promotion in Lycopersicon esculentum Mill and the treated plants also developed resistance against wilt pathogen, Fusarium oxysporum f. sp. lycopersici though the effect was well
pronounced with consortium developed from Santalum album.
Li, J., Q. Yang, et al. (2009). "Evaluation of biocontrol efficiency and security of A Bacillus subtilis strain B29 against cucumber Fusarium wilt in field." China Vegetables(2): 30-33.
cucumerinum, was isolated from cucumber rhizosphere. After twice of 4-field-plot experiments, the control efficiencies of 100, 250 and 500 dilution times to cucumber Fusarium wilt
were 70.3-88.2%, 62.3-85.9%, and 54.7-80.6%, respectively. The average efficiency of field trials with B29 was 84.9% during 2 years and the yield of cucumber increased by 12.57%.
The acute toxicity of Bacillus subtilis strain B29 to big mouse through its mouth and skin was examined, and the LD50 was more than 5000 mg/kg. The application of strain B29 on
cucumber, tomato, bean and seed pumpkin was safe based on the observed seedling rate, growth and development.
Liu, Q., J. C. Yu, et al. (2009). "Antagonism and Action Mechanism of Antifungal Metabolites from Streptomyces rimosus MY02." Journal of Phytopathology 157(5): 306-310.
The genus of Streptomyces, a saprophytic Gram-positive bacterium, has properties, which make them useful as pharmaceutical and biocontrol agents. A streptomyces strain MY02
from soil samples showed significant antagonism against 14 plant pathogenic fungi including Fusarium oxysporum f. sp. cucumarinum. Antifungal metabolite(s) SN06 from the culture
of the strain MY02 were extracted with n-butanol and purified by silica gel column chromatography. The minimum concentration of SN06 inhibiting any visible fungal growth of F.
oxysporum f. sp. cucumarinum is 12.5 mu g/ml by twofold serial dilutions method. The mycelia of F. oxysporum f. sp. cucumarinum treated with SN06 were observed under the
normal optics microscope. The results showed that some cells of hyphae began to dilate and formed some strings of beads. The cytoplasm oozed out of the cells with the culture time
and so most of the cells became empty. The hyphae broke into many segments and then collapsed after 48 h. After inoculated in potato dextrose medium for 48 h, the filtrate of
mycelia treated with 1% NaCl containing 12.5 mu g/ml SN06 was scanned using ultraviolet spectrophotometer and absorption peak at 260 nm showed that the mycelia cell membrane
of F. oxysporum f. sp. cucumarinum was broken and that nucleic acid oozed out of the cell.
Maina, M., R. Hauschild, et al. (2008). "Protection of tomato plants against fusaric acid by resistance induction." Journal of Applied Biosciences(JABs) 1: 18-31.
Objectives: The rhizobacteria Bacillus sphaericus B43, Pseudomonas fluorescens T58, and P. putida 53 are able to induce systemic resistance (ISR) against Fusarium oxysporum
f.sp. lycopersici (FOL) in tomato. This study investigated if the ISR reduced the damage by the toxin fusaric acid (FA) produced by FOL. Methodology and Results: The bacteria were
applied to the rhizosphere of tomato plants. Chlorophyll content and ion leakage were determined after placing the leaf discs in FA. Active oxygen species (AOS), superoxide and
hydrogen peroxide levels were determined in leaves of plants injected with FA. Activities of superoxide dismutase (SOD), ascorbate (AS) and guaiacol peroxidases (GPX) involved in
AOS metabolism were quantified. In untreated plants, FA led to high ion leakage and chlorophyll degradation caused by H2O2 accumulation. All the bacteria treatments decreased
the chlorophyll degradation. Ion leakage was reduced by treatment with P. fluorescens T58 and B. sphaericus B43, while P. putida 53 was less effective. Treatment of plants with
bacteria resulted in increased superoxide contents, but varying over time. Increased SOD and GPX activities in untreated plants were suppressed after bacteria treatment. Plants
treated with P. fluorescens T58 showed only a transient increase in superoxide. P. putida 53-treated plants removed AOS, but high initial superoxide levels led to membrane
damages. Treatment with B. sphaericus B43 suppressed the effects of FA, but AOS metabolism showed only slight alterations. Conclusions and potential applications of findings: ISR
could also protect plant tissues from damage by pathogen toxins, which is a potential new dimension to the known mechanisms of action of biological control agents.
Martinez-Medina, A., J. A. Pascual, et al. (2009). "Interactions between arbuscular mycorrhizal fungi and Trichoderma harzianum and their effects on Fusarium wilt in melon plants grown in
seedling nurseries." Journal of the Science of Food and Agriculture 89(11): 1843-1850.

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BACKGROUND: Biological control through the use of Trichoderma spp. and arbuscular mycorrhizal fungi (AMF) could contribute to a reduction of the inputs of environmentally
damaging agrochemical products. The objective of this study was to evaluate the interactions between four AMF (Glomus intraradices, Glomus mosseae, Glomus claroideum and
Glomus constrictum) and Trichoderma harzianum for their effects on melon plant growth and biocontrol of Fusarium wilt in seedling nurseries. RESULTS: AMF colonisation decreased
fresh plant weight, which was unaffected by the presence of T. harzianum. Dual inoculation resulted in a decrease in fresh weight compared with AMF-inoculated plants, except for G.
intraradices. AMF colonisation level varied with the AM endophyte and was increased by T. harzianum, except in G. mosseae-inoculated plants. Negative effects of AMF on T.
harzianum colony-forming units were found, except with G. intraradices. AMF alone were less effective than T. harzianum in suppressing disease development. Combined inoculation
resulted in a general synergistic effect on disease control. CONCLUSION: Selection of the appropriate AMF species and its combination with T. harzioanum were significant both in
the formation and effectiveness of AM symbiosis and the reduction of Fusarium wilt incidence in melon plants. The combination of G. intraradices and T. harzianum provided better
results than any other tested. (C) 2009 Society of Chemical Industry
Matar, S. M., S. A. El-Kazzaz, et al. (2009). "Antagonistic and inhibitory effect of Bacillus subtilis against certain plant pathogenic fungi, I." Biotechnology 8(1): 53-61.
subtilis isolates (B1 to B14), obtained from different Egyptian sites, were tested against six fungal isolates belonging to four different genera, Rhizoctonia solani, Helminthosporium
spp., Alternaria spp. and Fusarium oxysporum. Cultural, morphological and physiological characteristics of these isolates were found to be identical to B. subtilis. Four B. subtilis
isolates (B1, B4, B7, B8) had more antagonistic effect on all fungal isolates. Supernatant of B. subtilis isolate B7 had antagonistic effect on 6 fungal isolates but it was more effective on
Helminthosporium spp., Alternaria spp. and F. oxysporum. B. subtilis as well as isolate B7 showed effectiveness in reducing disease incidence and severity levels of tomato plants
when added to the F. oxysporum and R. solani-infested soil. Also, it stimulated the growth of tomato plants compared to the other. HPLC analysis of the HCl precipitate of B.subtilis
isolate B7 culture supernatant revealed that an identical pattern of five peaks to that of a purified preparation of iturin A was obtained.
Matar, S. M., S. A. El-Kazzaz, et al. (2009). "Bioprocessing and scaling-up cultivation of Bacillus subtilis as a potential antagonist to certain plant pathogenic fungi, III." Biotechnology 8(1):
138-143.
isolate G-GANA7 (GenBank accession No. EF583053), collected from Abo-Homos in Egypt, was tested against six fungal isolates belonging to four different genera, i.e. Rhizoctonia
solani, Helminthosporium sp., Alternaria sp. and Fusarium oxysporum. B. subtilis isolate G-GANA7 was cultured in 3 litre bench-top New Brunswick Scientific BioFlow III bioreactor for
producing the maximum yield of biomass and antifungal compound. Fed-batch processes were automated through a computer aided data bioprocessing system AFS-BioCommand
multi-process management program to regulate the cell growth rate by controlling interactively the nutrient feed rate, temperature, pH and agitation speed based on dissolved oxygen.
In batch cultivation, the process suffered from low yield of cell mass (3.2 g litre-1) and antifungal activity because of high initial glucose concentration followed by acetate formation
which the causal agent for inhibition of cell growth. Constant and exponential fed-batch strategies were adopted to circumvent this potential problem. Fed-batch cultivation of B. subtilis
was conducted at the specific growth rate of 0.13 and 0.1 h-1 for constant and exponential strategies, respectively. High cell density of 12.8 and 14.6 g litre-1 for both operations, with
an overall biomass yield of 0.45 g g-1 was achieved. The inhibitory activity of antifungal in supernatant reached its maximum value of 2 and 2.2 cm for constant and exponential fed-
batch cultivations.
Mazurier, S., T. Corberand, et al. (2009). "Phenazine antibiotics produced by fluorescent pseudomonads contribute to natural soil suppressiveness to Fusarium wilt." ISME Journal 3(8):
977-991.
Natural disease-suppressive soils provide an untapped resource for the discovery of novel beneficial microorganisms and traits. For most suppressive soils, however, the consortia of
microorganisms and mechanisms involved in pathogen control are unknown. To date, soil suppressiveness to Fusarium wilt disease has been ascribed to carbon and iron competition
between pathogenic Fusarium oxysporum and resident non-pathogenic F. oxysporum and fluorescent pseudomonads. In this study, the role of bacterial antibiosis in Fusarium wilt
suppressiveness was assessed by comparing the densities, diversity and activity of fluorescent Pseudomonas species producing 2,4-diacetylphloroglucinol (DAPG) (phlD+) or
phenazine (phzC+) antibiotics. The frequencies of phlD+ populations were similar in the suppressive and conducive soils but their genotypic diversity differed significantly. However,
phlD genotypes from the two soils were equally effective in suppressing Fusarium wilt, either alone or in combination with non-pathogenic F. oxysporum strain Fo47. A mutant
deficient in DAPG production provided a similar level of control as its parental strain, suggesting that this antibiotic does not play a major role. In contrast, phzC+ pseudomonads were
only detected in the suppressive soil. Representative phzC+ isolates of five distinct genotypes did not suppress Fusarium wilt on their own, but acted synergistically in combination with
strain Fo47. This increased level of disease suppression was ascribed to phenazine production as the phenazine-deficient mutant was not effective. These results suggest, for the first
time, that redox-active phenazines produced by fluorescent pseudomonads contribute to the natural soil suppressiveness to Fusarium wilt disease and may act in synergy with carbon
competition by resident non-pathogenic F. oxysporum.

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Minerdi, D., S. Bossi, et al. (2009). "Volatile organic compounds: a potential direct long-distance mechanism for antagonistic action of Fusarium oxysporum strain MSA 35." Environmental
Microbiology 11(4): 844-854.
Fusarium oxysporum MSA 35 [wild-type (WT) strain] is an antagonistic Fusarium that lives in association with a consortium of bacteria belonging to the genera Serratia,
Achromobacter, Bacillus and Stenotrophomonas in an Italian soil suppressive to Fusarium wilt. Typing experiments and virulence tests provided evidence that the F. oxysporum
isolate when cured of the bacterial symbionts [the cured (CU) form], is pathogenic, causing wilt symptoms identical to those caused by F. oxysporum f. sp. lactucae. Here, we
demonstrate that small volatile organic compounds (VOCs) emitted from the WT strain negatively influence the mycelial growth of different formae speciales of F. oxysporum.
Furthermore, these VOCs repress gene expression of two putative virulence genes in F. oxysporum lactucae strain Fuslat10, a fungus against which the WT strain MSA 35 has
antagonistic activity. The VOC profile of the WT and CU fungus shows different compositions. Sesquiterpenes, mainly caryophyllene, were present in the headspace only of WT MSA
35. No sesquiterpenes were found in the volatiles of ectosymbiotic Serratia sp. strain DM1 and Achromobacter sp. strain MM1. Bacterial volatiles had no effects on the growth of the
different ff. spp. of F. oxysporum examined. Hyphae grown with VOC from WT F. oxysporum f. sp. lactucae strain MSA 35 were hydrophobic whereas those grown without VOCs
were not, suggesting a correlation between the presence of volatiles in the atmosphere and the phenotype of the mycelium. This is the first report of VOC production by antagonistic F.
oxysporum MSA 35 and their effects on pathogenic F. oxysporum. The results obtained in this work led us to propose a new potential direct long-distance mechanism for antagonism
by F. oxysporum MSA 35 mediated by VOCs. Antagonism could be the consequence of both reduction of pathogen mycelial growth and inhibition of pathogen virulence gene
expression.
Nam, M. H., M. S. Park, et al. (2009). "Biological Control of Strawberry Fusarium Wilt Caused by Fusarium oxysporum f. sp fragariae Using Bacillus velezensis BS87 and RK1
Formulation." Journal of Microbiology and Biotechnology 19(5): 520-524.
Two isolates, Bacillus sp. BS87 and RK1, selected from soil in strawberry fields in Korea, showed high levels of antagonism towards Fusarium oxysporum f. sp. fragariae in vitro. The
isolates were identified as B. velezensis based on the homology of their gyrA sequences to reference strains. BS87 and RK1 were evaluated for control of Fusarium wilt in
strawberries in pot trials and field trials conducted in Nonsan, Korea. In the pot trials, the optimum applied concentration of BS87 and RK1 for pre-plant root-dip application to control
Fusarium wilt was 10(5) and 10(6) colony-forming units (CFU)/ml, respectively. Meanwhile, in the 2003 and 2005 field trials, the biological control efficacies of formulations of RK1
were similar to that of a conventional fungicide (copper hydroxide) when compared with a non-treated control. The RK1 formulation was also more effective than BS87 in suppressing
Fusarium wilt under field conditions. Therefore, the results indicated that formulations of B. velezensis BS87 and RK1 may have potential to control Fusarium wilt in strawberries.
Narayan, M., P. Tini, et al. (2009). "Biological and chemical management of tomato wilt caused by Fusarium oxysporum f.sp. lycopersici." Journal of Soils and Crops 19(1): 118-121.
Wilt of tomato is one of the most important known disease caused by Fusarium oxysporum f. sp. lycopersici. In the present study four bioagents (Trichoderma harzianum, T. viride,
Bacillus subtilis and Pseudomonas fluorescens) and two fungicides (Carbendazim and Thiram) were evaluated both in vitro and in vivo conditions. In vitro evaluation, of Carbendazim
(0.1%) completely inhibited the growth of tomato wilt pathogen Fusarium oxysporum f.sp. lycopersici and was found significantly superior over the rest of fungicides. While, among the
biological agents Trichoderma viride was found significantly superior to the rest in checking the growth of pathogens and showed 85.69 per cent inhibition. In vivo under field condition,
seedling dip treatment of Carbendazim (1 gl-1 water) was found most significant followed by Carbendazim+ T.viride (1+100 gl-1 water) and T. viride (100 gl-1 water) significantly
reduced wilt incidence by 73.91, 69.56 and 68.11 per cent respectively as against 71.88 per cent wilting in control (under epiphytotic condition i.e. wilt sick soil).
Ortega-Morales, B. O., F. N. Ortega-Morales, et al. (2009). "Antagonism of Bacillus spp. Isolated from Marine Biofilms Against Terrestrial Phytopathogenic Fungi." Marine Biotechnology
11(3): 375-383.
We aimed at determining the antagonistic behavior of bacteria derived from marine biofilms against terrestrial phytopathogenic fungi. Some bacteria closely related to Bacillus
mojavensis (three isolates) and Bacillus firmus (one isolate) displayed antagonistic activity against Colletotrichum gloeosporioides ATCC 42374, selected as first screen organism. The
four isolates were further quantitatively tested against C. gloeosporioides, Colletotrichum fragariae, and Fusarium oxysporum on two culture media, potato dextrose agar (PDA) and a
marine medium-based agar [yeast extract agar (YEA)] at different times of growth of the antagonists (early, co-inoculation with the pathogen and late). Overall antagonistic assays
showed differential susceptibility among the pathogens as a function of the type of culture media and time of colonization (P < 0.05). In general, higher suppressive activities were
recorded for assays performed on YEA than on PDA; and also when the antagonists were allowed to grow 24 h earlier than the pathogen. F. oxysporum was the most resistant
fungus while the most sensitive was C. gloeosporioides ATCC 42374. Significant differences in antagonistic activity (P < 0.05) were found between the different isolates. In general,
Bacillus sp. MC3B-22 displayed a greater antagonistic effect than the commercial biocontrol strain Bacillus subtilis G03 (KodiakA (R)). Further incubation studies and scanning

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electronic microscopy revealed that Bacillus sp. MC3B-22 was able to colonize, multiply, and inhibit C. gloeosporioides ATCC 42374 when tested in a mango leaf assay, showing its
potential for fungal biocontrol. Additional studies are required to definitively identify the active isolates and to determine their mode of antifungal action, safety, and biocompatibility.
Padghan, P. R. and M. M. Baviskar (2009). "Efficacy of bioagent and different root extracts for supression of chickpea wilt in vitro." Asian Journal of Bio Science 4(1): 56-58.
udid, sorghum (Sorghum bicolor), groundnut and mung bean and biological control agents (Trichoderma viride, T. harzianum, T lignorum and T. koningii) against the chickpea wilt
pathogen, Fusarium oxysporum f.sp. ciceris (FOC), was studied in the laboratory. A lower radial mycelial growth and a higher inhibitory effect were recorded in sorghum root extract
medium (28.00 mm and 54.34%), respectively, however, it was at par with groundnut root extract medium (30.00 mm and 51.08%), compared to the control (61.33 mm). In dual
culture technique, the growth of FOC was restricted by T. viride (56.16%), followed by T. harzianum (50.57%). T. lignorum recorded the minimum zone of inhibition (40.45%).
Qiu, W., H. Huang, et al. (2009). "Screening of actinomycete against Fusarium oxysporum f. sp. cubense and identification of strain DA07408." Research of Agricultural Modernization
30(1): 126-128.
samples, and 8 of these strains showed significant activities against F. oxysporum f.sp. cubense. One actinomycete (DA07408) isolated from an arboretum in Danzhou, Hainan,
China, exhibited marked antagonism towards F. oxysporum f.sp. cubense. The conditions for the fermentation of the actinomycete were optimized. Based on the morphological,
physiological and biochemical characteristics of the strain, and on the analysis of 16S rDNA and phylogenetic tree, DA07408 was identified as Streptomyces olivochromogenes.
Raddadi, N., A. Belaouis, et al. (2009). "Characterization of polyvalent and safe Bacillus thuringiensis strains with potential use for biocontrol." Journal of Basic Microbiology 49(3): 293-303.
Sixteen Bacillus thuringiensis (Bt) strains were screened for their anti-insect, antibacterial and antifungal determinants by phenotypic tests and PCR targeting major insecticidal
proteins and complements, chitinases, lactonases, beta-1,3-glucanases and zwittermicin A. Six strains had genes of at least two major insecticidal toxins and of insecticidal
complements. With regard to fungal biocontrol, all the strains inhibited Fusarium oxysporum and Aspergillus flavus growth and four strains had all or most of the antifungal
determinants examined, with strain Bt HD932 showing the widest antifungal activity spectrum. Autolysins, bacteriocin and AHL-lactonases were produced by all or most of the tested
strains with different activity spectra including pathogens like Listeria monocytogenes. Safety evaluation was carried out via PCR by screening the B. cereus psychrotolerance-related
genes, toxin genes and the virulence pleiotropic regulator plcR. Diarrheal enterotoxins and other toxin genes were widespread among the collection with strains Bt HD9 and H45
lacking psychrotolerance-related genes, while five strains were positive. Only three strains (BMG1.7, H172, H156) resulted positive with primer sets targeting partial or complete plcR
gene. By Vero Cell Assays, Bt HD868 followed by Bt HD9 were shown to be the safest strains. These polyvalent and safe Bt strains could be very promising in field application.
Rasal, P. H., J. R. Shelar, et al. (2009). "Effect of endophytic antagonist on pigeonpea." Journal of Maharashtra Agricultural Universities 34(1): 52-53.
resistant (ICP 8863) and resistant (BDN2) cultivars of pigeon pea were screened against Fusarium oxysporum f. udum [F. udum]. The inoculation of endophytic antagonists into
different cultivars of pigeon pea improved germination, plant height, branching, nodulation, root length and biomass production, and reduced wilt intensity significantly over the un-
inoculated control. Among the inoculants, Pseudomonas-2 was the most beneficial, followed by Pseudomonas-3, Bacillus-3, Pseudomonas-1, and Bacillus-1 and -2. Antagonists
isolated from resistant cultivar were the most beneficial, followed by antagonists from the moderately resistant cultivar, and antagonists isolated from the susceptible cultivar.
Recep, K., S. Fikrettin, et al. (2009). "Biological control of the potato dry rot caused by Fusarium species using PGPR strains." Biological Control 50(2): 194-198.
In this study, a total of 17 Plant Growth Promoting Rhizobacteria (PGPR) strains, consisting of eight different species (Bacillus subtilis, Bacillus pumilus, Burkholderia cepacia,
Pseudomonas putida, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus macerans and Flavobacter balastinium), were tested for antifungal activity in in vitro (on Petri plate) and
in vivo (on potato tuber) conditions against Fusarium sambucinum, Fusarium oxysporum and Fusarium culmorum cause of dry rot disease of potato. All PGPR strains had inhibitory
effects on the development of at least one or more fungal species on Petri plates. The strongest antagonism was observed in B. cepacia strain OSU-7 with inhibition zones ranging
from 35.33 to 47.37 mm. All PGPR strains were also tested on tubers of two potato cultivars 'Agria' and 'Granola' under storage conditions. Only B. cepacia strain OSU-7 had
significant effects on controlling potato dry rot caused by three different fungi species on the two potato cultivars. There were no significant differences in rot diameters among the
treatments in comparison to the negative control (with water). This is the first study showing that B. cepacia has great potential to be used as effective biocontrol agent of Fusanium dry
rot of potatoes (F. oxysporum and F culmorum) under storage conditions. (C) 2009 Elsevier Inc. All rights reserved.
Riaz, T., S. N. Khan, et al. (2009). "Effect of co-cultivation and crop rotation on corm rot disease of Gladiolus." Scientia Horticulturae 121(2): 218-222.

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Field and pot experiments were conducted to evaluate the effect of co-cultivation and crop rotation on the growth and corm rot disease of gladiolus (Gladiolus grandiflorus sect.
Blandus) cv. Aarti caused by Fusarium oxysporum f.sp. gladioli (Massey) Snyd. and Hans. In the field experiment, gladiolus was co-cultivated with 10 agricultural/horticultural crops
viz. Allium cepa L., Brassica campestris L., Capsicum annuum L., Eruca sativa Mill., Helianthus annuus L., Tagetes erectus L., Zea mays L., Vinca rosea L. and Rosa indica L., in a
soil infested with F. oxysporum. All the crops except V. rosea and R. indica reduced disease incidence. The effect of H. annuus and T. erectus was significant and more pronounced
than other co-cultivated crops. In general, root and shoot dry biomass, corm fresh weight, number of cormlets and number of flowers per spike decreased as compared to the un-
inoculated monoculture gladiolus treatment (negative control) but these parameters enhanced as compared to the F. oxysporum inoculated monoculture gladiolus treatment (positive
control). In a pot experiment, all the crops of the field experiment except V. rosea and R. indica were sown in rotation with gladiolus. Pot grown plants of different species were
harvested at maturity and the soil was inoculated with F oxysporum. Gladiolus was cultivated I week after inoculation. Disease incidence was significantly suppressed in all the
treatments ranging from 29% to 53%. The highest suppression of disease incidence was recorded in T erectus (53%) followed by B. campestris (49%). The effect of preceding crops
on various vegetative parameters was similar in the pot experiment to that of the field experiment. The present study suggests that corm rot disease of gladiolus can be managed by
mixed cropping of H. annuus and T erectus or cultivation of T. erectus and B. campestris in rotation. (c) 2009 Elsevier B.V. All rights reserved.
Saidi, N., S. Kouki, et al. (2009). "Characterization and selection of Bacillus sp strains, effective biocontrol agents against Fusarium oxysporum f. sp radicis-lycopersici, the causal agent of
Fusarium crown and root rot in tomato." Annals of Microbiology 59(2): 191-198.
The antagonistic activities of 20 Bacillus isolates were tested with dual culture and greenhouse conditions against Fusarium oxysporum f. sp. radicis-lycopersici (FORL) race 0, the
causal agent of Fusarium crown and root rot of tomato. Under dual culture, 10 isolates inhibited mycelial growth > 38% and the most effective inhibited fungal growth > 50%. The 20
Bacillus isolates were tested for production of volatiles, cyanide, antibiotics, and phosphorus solubilisation; 15 isolates produced volatiles that inhibited growth of pathogens, 9 isolates
produced cyanide, 10 produced antibiotics, and five solubilised phosphorus. Greenhouse experiments with the same 20 isolates revealed the effectiveness of 12 strains, which
increased the percentage of healthy plants in the tested cultivar from 66 to 96%. The best disease control was achieved by isolates B11, B5, B17, and B18. However, B11 and B17
were the only isolates that produced cyanide, antibiotics, solubilised phosphate and showed 44% inhibition of fungal growth. The selected strains could be considered in plant growth
promotion and biological disease control.
Shi, Y. W., K. Lou, et al. (2009). "Isolation, quantity distribution and characterization of endophytic microorganisms within sugar beet." African Journal of Biotechnology 8(5): 835-840.
The present investigation was undertaken in order to document the spectrum of endophytes colonizing healthy leaves of sugar beet cultivars in Xinjiang Province ( China) and to
determine the degree of colonization at three growth stages. From the 360 sugar beet leaf and root segments incubated, 221 bacterial isolates, 34 fungal isolates and 5 actinomycete
isolates were obtained. Of all the isolates, 7 bacterial species and 6 fungal species were identified. The actinomycete isolates were characterized as Streptomyces griseofuscus and
Streptomyces globisporus. There were significant differences between microorganisms, stages of growth, and stages of microorganism interaction. The number of microorganisms
isolated increased during the growth period of the sugar beet. At the same time, the number of microorganisms affecting different parts of the sugar beet tissue was quite different. The
greatest number of microorganisms was found in the secondary root emergence zone of the sugar beet tissue. Endophytic microorganisms in sugar beet promote growth and
increase the yield of the beet.
Son, S. H., Z. Khan, et al. (2009). "Plant growth-promoting rhizobacteria, Paenibacillus polymyxa and Paenibacillus lentimorbus suppress disease complex caused by root-knot nematode
and fusarium wilt fungus." Journal of Applied Microbiology 107(2): 524-532.
Paenibacillus strains against disease complex caused by Meloidogyne incognita and Fusarium oxysporum f. sp. lycopersici interactions. Methods and Results: Paenibacillus strains
were collected from rotten ginseng roots. The strains were tested under in vitro and pots for their inhibitory activities, and biocontrol potential against disease complex caused by M.
incognita and F. oxysporum f. sp. lycopersici on tomato. In in vitro experiments, among 40 tested strains of Paenibacillus spp., 11 strains showed antifungal and nematicidal activities
against F. oxysporum f. sp. lycopersici and M. incognita, respectively. Paenibacilluspolymyxa GBR-462; GBR-508 and P. lentimorbus GBR-158 showed the strongest antifungal and
nematicidal activities. These three strains used in pot experiment reduced the symptom development of the disease complex (wilting and plant death), and increased plant growth.
The control effects were estimated to be 90-98%, and also reduced root gall formation by 64-88% compared to the untreated control. Conclusion: The protective properties of selected
Paenibacillus strains make them as potential tool to reduce deleterious impact of disease complex plants. Significance and Impact of the Study: The study highlights biocontrol
potential of Paenibacillus strains in management of disease complex caused by nematode-fungus interaction.

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Srinivasan, K., G. Gilardi, et al. (2009). "BACTERIAL ANTAGONISTS FROM USED ROCKWOOL SOILLESS SUBSTRATES SUPPRESS FUSARIUM WILT OF TOMATO." Journal of
Plant Pathology 91(1): 147-154.
Five bacterial E,trains (FC-6B, FC-7B, FC-8B, FC-9B and FC-24B) isolated from used rockwool soilless substrates were identified using 16S ribosomal DNA (16S rDNA) sequence
analysis as belonging to the Pseudomonas genus. Seven glasshouse trials were conducted in order to evaluate the efficacy of these bacteria strains (Pseudomonas putida FC-6B,
Pseudomonas sp. FC-7B, Pseudomonas putida FC-8B, Pseudomonas sp. FC-9B and Pseudomonas sp. FC-24B) together with Achromobacter sp. AM1 and Serratia sp. DM1
obtained from suppressive sod, against Fusarium wilt of tomato. Two commercial bioproducts, Trichoderma harzianum T22 (RootShield) and Pseudomonas chlororaphis MA 342
(Cedomon) were also evaluated. Different treatment strategies including soil application (10(7) and 10(8) cfu ml(-1)) were adopted in different glasshouse trials (Trial I to VI) to test the
efficacy of the bacterial strains against Fusarium wilt. Root dipping was used in Trial VII (10(8) and 10(9) cfu ml(-1)). The lowest: disease incidence (3.3) was recorded with a single
application of P. putida FC-6B at 10(8) cfu ml(-1). Similar results were obtained with the same bacteria when the concentration was decreased to 10(7) cfu ml(-1) but an increasing
number of applications was required. The highest plant biomass (50.3 g/plant) was recorded in the P. putida FC-8B treatment (Trial III). In conclusion, the current study showed the
potential biocontrol activity of bacterial strains FC-6B, FC-7B, FC-8B, FC-9B and FC-24B isolated from re-used rockwool soilless substrates against Fusarium wilt disease, and the
growth promoting activity of these strains on tomato plants.
Srivastava, D. K., A. K. Singh, et al. (2009). "Efficacy of bio-control agents and seed dressing fungicides against damping off of tomato." Annals of Plant Protection Sciences 17(1): 257-
258.
in Unao, Madhya Pradesh, India, during 2005-06 yielded associated pathogen on PDA medium. The antagonistic activity of biological control agents against Fusarium oxysporum f.sp.
lycopersici was determined using dual culture method. All the antagonists and fungicide inhibited the mycelial growth of Fusarium, however, Trichoderma viride caused maximum
inhibition of mycelial growth. Trichoderma viride, Trichoderma harzianum, Gliocladium virens, carbendazim and thiram, which showed significant in vitro inhibition of Fusarium were
tested in the field. Maximum increase in seed germination (83.4%), seedling survival (79.0) and plant height (6.32 cm) over the control was observed when treated with Trichoderma
viride followed by Trichoderma harzianum, carbendazim, thiram, and Gliocladium virens.
Thanh, D. T., L. T. T. Tarn, et al. (2009). "Biological Control of Soilborne Diseases on Tomato, Potato and Black Pepper by Selected PGPR in the Greenhouse and Field in Vietnam." Plant
Pathology Journal 25(3): 263-269.
Bacterial wilt, Fusarium wilt and Foot rot caused by Ralstonia solanacearum, Fusarium oxysporum, and Phytophthora capsici respectively, continue to be severe problems to tomato,
potato and black pepper growers in Vietnam. Three bio-products, Bacillus vallismortis EXTN-1 (EXTN-1), Bacillus sp. and Puenibacillus sp. (ESSC) and Bacillus substilis (MFMF)
were examined in greenhouse bioassay for the ability to reduce bacterial wilt, fusarium wilt and foot rot disease severity. While these bio-products significantly reduced disease
severities, EXTN-1 was the most effective, providing a mean level of disease reduction 80.0 to 90.0% against bacterial wilt, fusarium wilt and foot rot diseases under greenhouse
conditions. ESSC and MFMF also significantly reduced fusarium wilt, bacterial wilt and foot rot severity under greenhouse conditions. Bio-product, EXTN-1 with the greatest efficacy
under greenhouse condition was tested for the ability to reduce bacterial wilt, fusarium wilt and foot rot under field condition at Song Phuong and Thuong Tin locations in Ha Tay
province, Vietnam. Under field condition, EXTN-1 provided a mean level of disease reduction more than 45.0% against all three diseases compared to water treated control. Besides,
EXTN-1 treatment increased the yield in tomato fruits 17.3% than water treated control plants.
Wu, H., X. Yang, et al. (2009). "Suppression of Fusarium wilt of watermelon by a bio-organic fertilizer containing combinations of antagonistic microorganisms." BioControl 54(2): 287-300.
the crop has been grown for many seasons. Its occurrence results in a severely decreased watermelon crop. The goal of this study was to assess the capability of a new product (bio-
organic fertilizer) to control the wilt in Fusarium-infested soil. Pot experiments were conducted under growth chamber and greenhouse conditions. The results showed that the fertilizer
controlled the wilt disease. Compared with control pots, the incidence rates of Fusarium wilt at 27 and 63 days following treatment of the plants with the bio-organic fertilizer at a rate of
0.5% (organic fertilizer+antagonistic microorganisms, including 3*109 CFU g-1 respectively, in both the growth chamber and greenhouse settings. The activities of antioxidases
(catalase, superoxide dismutase and peroxidase) in watermelon leaves increased by 38.9, 150 and 250%, respectively. In the roots, stems and leaves, the activity of beta-1,3-
glucanase (pathogenesis-related proteins) increased by 80, 1140 and 100% and that of chitinase increased by 240, 80, and 20%, respectively, while the contents of malondialdehyde
fell by 56.8, 42.1 and 45.9%, respectively. These results indicate that this new fertilizer formula is capable of protecting watermelon from Fusarium oxysporum f.sp. niveum. The
elevated levels of defense-related enzymes are consistent with the induction and enhancement of systemic acquired resistance of plant.

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Wu, Q., H. Zeng, et al. (2009). "Stability of fermentation broth of actinomycete strain WZ162 resistance to Fusarium oxysporum f.sp. cubense of banana." Guangxi Agricultural Sciences
40(4): 366-369.
The fermentation broth of actinomycete strain WZ162 has strong inhibiting effect against Fusarium oxysporum f.sp. cubense of banana. Under different conditions, the stabilities of
fermentation broth of WZ162 were detected. The results showed that the fermentation broth of WZ162 had better heat stability when temperature of water bath was below 80C. The
antibiotics ingredient of fermentation broth would not be changed and can maintain the antifungal activity under conditions of sun light and ultraviolet rays. Under acid and neutrality
conditions, the inhibition rate of fermentation broth against Focr4 was 24.92%-34.73% and 11.21%-25.39%, respectively. Therefore, the stability of fermentation broth in acid was
better than that of neutrality. When the fermentation broth with pH 1-12 were treated with different time in 100C water bath, the inhibition rate was obviously lower than that of the
treatments without water bath, and the stability of fermentation broth with pH 1 was the best.
Yin, X., D. Chen, et al. (2009). "An endophytic Erwinia chrysanthemi strain antagonistic against banana fusarium wilt disease." Chinese Journal of Biological Control 25(1): 60-65.
An endophytic strain E353 was obtained from the pseudostem of healthy banana plant in a field heavily infected with Fusarium oxysporum f. sp. cubense (FOC). Antagonism of the
strain against FOC was tested via dual-culture, inhibition test on conidia germination, and pot trials. Results showed that E353 effectively inhibited mycelium growth and conidia
germination. Efficacy of strain E353 to control the wilt disease was 60.67% in pot trials. Strain E353 was identified as Erwinia chrysanthemi according to its characteristics in
morphology, physiology, biochemistry and 16S rDNA sequence.
Zhong, X., M. Liang, et al. (2009). "Study on the inhibition of Trichoderma sp. against Fusarium oxysporum f. sp. cubense in banana." Journal of Fruit Science 26(2): 186-189.
effective antagonist against Fusarium oxysporum f. sp. cubens, was isolated and identified as Trichoderma sp. based upon 18S rDNA gene analysis. With solid and liquid cultures, the
inhibitive efficacy to the growth of Fusarium oxysporum f. sp. cubens was primarily studied. The experimental results showed that the cells of Fusarium oxysporum f. sp. cubens were
completely covered by short fiber-like hyphace and spore stem of G2 within 7 days in the dual culture plate, and in the antagonist plate, the average rate of inhibitory by the culture
solution of G2 was about 90.4%, the average rate of the inhibitory by volatile substance reached 68.3%. After 10 days' incubation with 20% (v/v) fungal strain G2, the melt of the
pathogenic mycel and spore were observed in the liquid culture containing 1.0*107 cfu . L-1 G2 can strongly inhibit the growth of Fusarium oxysporum f. sp. cubens.
Zhu, H., Y. Ma, et al. (2009). "Control effect of combining biocontrol strains against Fusarium oxysporium f. sp. niveum and Verticillium dahliae." Journal of Northwest A & F University -
Natural Science Edition 37(7): 152-156.
Objective: Five actinomycetes strains having certain inhibiting capability were screened as material to study the control effect of the actinomycetes and five combinations on
watermelon Fusarium wilt and Eggplant Verticillium wilt, and to filter the combining biocontrol strains which have better biocontrol efficacy and growth promotion. Method: The
biocontrol efficacy and growth promotion of single and combining strains were analyzed by antagonistic activity in vitro and manual inoculation in vivo. Result: Strain SC11 and SE2
had significant inhibiting effect on Fusarium oxysporium f. sp. niveum and Verticillium dahliae in vitro. Inhibiting rate on conidia germination was also high; in greenhouse experiment,
84.93% control ratio to Fusarium oxysporium f. sp. niveum and 71. 48% to Verticillium dahliae were found by C2; The fermentation broth of C3 had the most significant effect for every
index of watermelon. The effect on reduction intensity of watermelon rootage was obvious. For eggplant, the growth promotion was only inferior to strain SF6. Conclusion: These
results suggested that the control effect and growth promotion of combining biocontrol strains are significantly higher than individual, and combining strains express complementary
biocontrol activities by collaboration. There is no correlation between the number of strains and control effect, only proper combinations of biocontrol strains can enhance disease
control effect.

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Kirchmair et al.,
2007
Metarhizium anisopliae Daktulosphaira
vitifoliae
(grape phylloxera)
Hemiptera:
Phylloxeridae
Germany Inundative Field + 3 months after application an increase
of the M.a. density in soil was observed.
Compared with untreated plots a lower
infestation was observed in the M.a.-
treated plots. Two years after treatment
a control effect was still observed
whereas the density of M.a. in soil
decreased. Three years after treatment
no effect on the pest was detectable
and the M.a. density had decreased to
a value similar to that in the control . A
periodically application is necessary.
Maheshkumar-
Katke & Balikai,
2008
Metarhizium anisopliae,
Verticillium lecanii,
Clerodendron inerme
Maconellicoccus
hirsutus
(grape mealybug)
Hemiptera:
Pseudococcidae
India Inundative Field +
* + means effective, - means not effective biocontrol agent.
17.6 Nematodes [5 species]
Reference Species of biocontrol
agent
Species of insect
pest
Taxonomic
category of
pests
Country Type of
augmentation
Type
of test
Efficacy of
biocontrol
agents*
Additional information and
results
Saunders & All,
1985
Steinernema carpocapsae Vitacea polistiformis
(grape root borer)
Lepidoptera:
Sesiidae
Georgia,
USA
Inundative
(soil)
Lab,
Field
+ Susceptibility of V.p. 1st-instar
larvae. Augmentation of nematode
populations during the critical
period of V.p. oviposition and
eclosion is suggested as a control
technique.
English-Loeb et
al., 1999
Heterorhabditis
bacteriophora
(Oswego strain),
Steinernema glaseri
(isolate 326)
Daktulosphaira
vitifolia
(grape phylloxera -
root form)
Hemiptera:
Phylloxeridae
NY,
USA
Lab +
-
-
H. bacteriophora: reduced survival
of attached phylloxera by up to
80%.
S. glaseri had no measurable
impact.
No evidence that H.b. could
successfully reproduce within the
bodies of the hosts.
Augmentative use in the field in an
release programme may be
constrained by the need to use
high densities, their dependence on
moist soils, and their inability to
propagate themselves within hosts.